Synthesis method of protein with modified profile of n-glycosylation in plants

FIELD: biotechnologies.

SUBSTANCE: method involves introduction to a plant, some part of the plant or a plant cell of nucleotide sequence for 80-100% of identical nucleotide sequence determined in SEQ ID NO: 17, and coding a composite protein containing a cytoplasmic end segment, a transmembrane domain, a steam area (CTS domain) of N-acetylglucosaminyl transferase (GNT1), which is merged with catalytic domain of beta-1,4-galactosyl transferase (GalT); with that, the above first nucleotide sequence is functionally connected to the first regulatory area being active in the plant; and the second nucleotide sequence for coding of a target protein; with that, the above second nucleotide sequence is functionally connected to the second regulatory area being active in the plant, as well as transient co-expression of the first and the second nucleotide sequences with synthesis of the target protein containing glycans, with reduced xylosylation, reduced fucosylation or their combination at comparison to the same target protein obtained from a wild plant. The invention described nucleic acid coding the protein that modifies glycosylation of target protein, a composite protein for modification of glycosylation of target protein; nucleic acid that codes it, as well as a plant, a plant cell and a seed, which contain the above nucleic acid or the above composite protein.

EFFECT: invention allows effective production of a target protein with reduced xylosylation, reduced fucosylation or their combination.

20 cl, 7 dwg, 9 ex

 

The technical FIELD TO WHICH the INVENTION RELATES.

The present invention relates to methods modified production of glycoproteins in plants. The present invention also involves the use of plants with modified production of glycoproteins.

BACKGROUND of INVENTION

Immunoglobulins (IHH) is a complex heteropolymer proteins with a characteristic affinity for the specific antigen analogues of different nature. Currently, regular isolating cell lines, forming the IHH, and the emergence of technology-oriented IHH evolution and molecular engineering has seriously affected their development as biotherapy and General market products for biomedical research. Therapeutic monoclonal IHH (monoclonal antibodies (MAB)to dominate an existing market of new anti-inflammatory and anti-cancer therapies. And now hundreds of new potential therapeutic drugs are at the stage of research and clinical trials to improve their properties or to define new directions for their use. The annual market demand µa ranges from a few grams (diagnosis), several kilograms (antivenoms) to one or several hundred kilograms (biosecurity, protivorakovye, anti-infective, anti-inflammatory drugs).

Despite the fact that the cultivation of cells SNO continues to be the preferred primary production on an industrial scale, it is generally accepted that in order µa reached its full impact on the market of products of biomedical research, it is necessary to create a backup of the production capacity. Since the power required for the production of these crops cannot be easily reconstructed in scale, the cost of their construction and maintenance are too high and constantly increasing, and their legalization in accordance with the rules of the organization of production and quality control of medicinal products (GMP) still takes on average three years after their creation. Even in the early stages of the selection cell lines Cho with an acceptable result at the output and efficiency of production remains expensive and lengthy process. The new facility, which will reduce the growing costs (higher yield, simpler technology and infrastructure), have a shorter time of commissioning, are easily sharable, while compliance with current properties, reproducibility, quality and safety in circulation systems cellular Kul is ur likely to significantly influence the development of ICA and vaccines to market the products of biomedical research at each stage of development.

Plants are easy "masters" for MCA and other proteins currently used in biological research (see Ko and Koprovski, 2005; MA et al., 2005; Yusibov et al., 2006 for reviews of the latest results). Receiving MCA was in stable lines of transgenic plants with an output of up to 200 mg/kg wet weight (CM) and by transient expression in amounts up to 20 mg/kg / CM (Kathuria, 2002). The report Giritch et al. (2006) mentioned the levels of expression of the magnitude of 200-300 mg/kg leaf mass for IHH, one of the mentioned levels exceed 500 mg/kg, achieved through the use of transition expression.

Process N-glycolysation in plants and mammals is different. The last steps N-glycolysation in the mammalian cell attached β1,4 galactose α1,6 fucose (beta-1,4 galactose, alpha,6 fucose and sialic acid residues to the complex picenum. However, in the plant residues are added β1,3 galactose α1,3 fucose (beta-1,3 galactose, alpha,3 fucose), α1,4 fucose and β1,2 xylose (alpha,4 fucose and beta,2 xylose). Alpha,3 fucose and beta,2 xylose, are components of Glyco-epitopes of a number of plant allergens. These residues are considered to be potentially immunodeficiency is ogenyi (allergenic), and their appearance in therapeutic proteins, including antibodies, are not considered desirable.

The addition of KDEL sequence to the C-end of the peptide is typically used to ensure extraction of the peptide from cells correlates with its return to ER (endoplasmic by reticulum). This approach was applied to obtain neuchateloisependule and necrotelicomnicon antibodies using agroinfiltration tobacco leaves (Sriraman et al., 2004). However, attached KDEL-peptide is a potential allergen, so this approach is of limited use in production of therapeutic proteins.

The control accession α1,3 fucose (alpha,3 fucose and β1,2 xylose (beta,2 xylose) was also achieved by modifying the expression of fucosyltransferase and xylosyltransferase. Mutants unable to attach α1,3 fucose and β1,2-xylose to complex picenum, were obtained from moss (Physcomitrella patens; Koprivova et al., 2004) and Arabidopsis thaUana (Strasser et al., 2004). Partial inhibition of the expression of fucosyltransferase and xylosyltransferase plants was also achieved through the expression of RNA inhibition (RNA/focused on genes α1,3 fucosyltransferase and β1,2 xylosyltransferase in Lemna minor Cox et al., 2006). However, complete inhibition of this enzymatic activity has a deleterious effect on some species, because of the and interfere with the normal flow key, development-related events, such as the formation of pollen or tying seeds. Caused by RNA-inhibition-specific degradation of mRNA (messenger RNA) may not remain stable for a long time, and it cannot be applied to a wide range based on the plants of the platforms used for medicinal drugs, since, as reported in the report, it is sensitive to the effects of environmental factors.

Patent WO 03/078637 contains a description of the application galactosyltransferase person to accelerate accession final β1,4-galactose (GalT) to picenum plants. Expression of galactose and the direction of its action on the CIS-Golgi by using synthesis with the transmembrane domain xylosyltransferase led to the joining end of galactose and reduction of specific residues in the plant, the raw N-glikana (see also Bakker et al., 2006). The cultivation of such plants containing recombinant IHH, has led to a significant, but variable reduction glycans comprising fucose and xylose.

Attaching the remainder of the beta-1,4-linked N-acetylglucosamine (GlnANc) to the beta-linked-mannose to get divided in half GlcNac accelerated N-acetylglucosaminyltransferase III (GnT-III; EC 2.4..1.144). Introduction this enzyme has been described Rouwendal et al. in 2007, plants, expressyou the proteins, contained GnT-III complex N-glycanase, were divided in half and carried two balance GlnAc.

SUMMARY of the INVENTION

The present invention relates to a method of modifying the production of glycoproteins in plants. The present invention also provides plants with modified production glycoprotein.

The aim of the present invention is to propose an improved method for modifying the formation of glycoproteins in plants.

Features nucleic acid having the nucleotide sequence of (A)containing nucleotides 1-1077 sequence SEQ ID NO. 17 (GNT1-GalT; fig.5d) or containing the nucleotide sequence showing from 80 to 100% identity with nucleotides 1-1077 sequence SEQ ID NO. 17, which is confirmed by using the following parameters: Program: blastn; Database: number; Expect 10; filter: low complexity; Location: in pairs; word Size: 11, where the nucleotide sequence encodes a protein that modifies glycosylation of a target protein.

Also features nucleic acid having the nucleotide sequence of (C)containing the first sequence of nucleic acid having the nucleotide 5-1198 sequence SEQ ID NO. 14 (GalT), or containing the nucleotide sequence, d is monsterous from 80 to 100% identity with nucleotides 5-1198 sequence SEQ ID NO. 14, which is confirmed by using the following parameters: Program: blastn; Database: number; Expect 10; filter: low complexity; Location: in pairs; word Size: 11, where the first nucleic acid sequence encodes a protein that modifies glycosylation of a target protein, and the first sequence of nucleic acid is functionally linked to the second nucleic acid sequence containing 35S, or plastocyanin, the promoter.

The present invention describes a nucleic acid having the nucleotide sequence containing the nucleotide 1-1641 sequence SEQ ID NO. 26 (GNT1-GnT-III) or containing the nucleotide sequence showing from 80 to 100% identity with nucleotides 1-1641 sequence SEQ ID NO. 26, which is confirmed by using the following parameters: Program: blastn; Database: number; Expect 10; filter: low complexity; Location: in pairs; word Size: 11, where the nucleotide sequence encodes a protein that modifies glycosylation of a target protein.

Also described are nucleic acid having the nucleotide sequence containing the first sequence of nucleic acid having the nucleotide 1-1460 sequence SEQ ID NO. 16 (GnT-III) or containing the nucleotide sequence, demonstrating the Yu from 80 to 100% identity with nucleotides 1-1460 sequence SEQ ID NO. 16, which is confirmed by using the following parameters: Program: blastn; Database: number; Expect 10; filter: low complexity; Location: in pairs; word Size: 11, where the first nucleic acid sequence encodes a protein that modifies glycosylation of a target protein, and the first sequence of nucleic acid is functionally linked to the second nucleic acid sequence containing 35S, or the promoter of plastocyanin.

In addition, we offer a plant, plant cell, seed, containing the nucleotide sequence of (A), (B), (C) or (D)described above.

The present invention also relates to a composite (hybrid) protein GNT1-GalT, containing CTS domain of N-acetylglucosaminyltransferase, fused with the catalytic domain of the beta 1,4 galactosyltransferase, and contains the sequence SEQ ID NO. 18. Amino acid sequence of sequence SEQ ID NO. 18 may be encoded by nucleotide sequence SEQ ID NO. 17.

Also features a plant, plant cell or seed containing the just-mentioned composite protein. In addition, we offer a plant, plant cell or seed containing nucleic acid having the sequence SEQ ID NO. 17.

The present invention includes a compound (hybrid) white is GNT1 - GnT-III, containing CTS domain of N-acetylglucosaminyltransferase, fused with the catalytic domain of N-acetylglucosaminyltransferase III, and also contains the amino acid sequence of SEQ ID NO. 20, the Amino acid sequence of sequence SEQ ID NO. 21 may be encoded by nucleotide sequence SEQ ID NO. 26.

Also features a plant, plant cell or seed containing the just-mentioned composite protein. In addition, we offer a plant, plant cell or seed containing nucleic acid having the nucleotide sequence SEQ ID NO. 26.

In accordance with the present invention it is proposed a method (1) for the synthesis of the target protein containing the expression within plants or parts of plants, the nucleotide sequence encoding a first nucleotide sequence encoding a protein compound GNT1-GalT, containing CTS domain of N-acetylglucosaminyltransferase (GNT1), fused with the catalytic domain of the beta 1,4 galactosyltransferase (GalT), the first nucleotide sequence is functionally associated with a first regulatory region that is active in the plant, and the second nucleotide sequence for encoding the target protein. The second nucleotide sequence is functionally linked to a second regulatory region, which is active in the plant, and the expression of the first and second nucleotide sequences to synthesize the target protein containing glikana with modified N-glycosylation.

As stated above, the first nucleotide sequence and the second nucleotide sequence can be expressed in the plant is unstable, or they can be expressed stably. Next, the first regulatory region may be the first tissue-specific promoter, and a second regulatory region is the second tissue-specific promoter. Both the first and second promoters may be promoters of plastocyanin.

The present invention also proposes a method (2) for the synthesis of the target protein. This method is similar to method 1, the target protein can be an antibody. If the target protein is an antibody, the second nucleotide sequence encoding a target protein, contains the nucleotide sequence 2A, functionally associated with the regulatory area 2A, which is active within the plant, and the second nucleotide sequence 2B, functionally associated with the regulatory area 2C that is active in the plant, and the product encoded by each of the sequences 2A and 2B are combined to produce the protein. Regulatory area 2A may be the promoter of plastocyanin, while the regulatory area 2B can also be a promoter of plastocyanin.

The present invention also features the above method (1) or method (2), where the third nucleotide sequence is expressed in the plant. A third nucleotide sequence encoding a suppressor of silencing (suppressor mechanism to "turn off" genes), functionally connected with the third regulatory region that is active in the plant. A third nucleotide sequence that encodes a suppressor of silencing, may be, for example, HcPro, TEV-p1/HC-Pro? BYV-p21, TBSV-p19, TCV-CP, CMV-2b, PVX-p25, PVM-p11, PVS-p11, BScV-p16, CTV-p23, GLRaV-2 p24, GBV-p14, HLV-p10, GCLV-p16 or GVA-p10. The third tissue promoter can be a promoter of plastocyanin.

In the present invention proposes a system for the expression of a plant for drying of expression of a target protein in a plant, where the target protein contains a modified glycosylation pattern. For example, the target protein contains reduced fokusirovannyi, xylopyranose or simultaneously fokusirovannyi and xylopyranose N-glikana. Alternatively, the target protein can contain a modified glycosylation pattern, wherein the protein is missing fokusirovannyi, xylopyranose or simultaneously fokusirovannyi and xylopyranose residues and the protein shows ovalicin the s Galatasaray. In addition, there could be an accession of terminal galactose, which will lead to the reduction or disappearance of fokusirovanie and xylotriose target protein when compared with the same target protein obtained in the wild plant.

In the present invention proposes a method (3) for the synthesis of the target protein with a modified profile of the N-glycosylation containing coexpressed within the plant, plant part or plant cell a nucleotide sequence that encodes a first nucleotide sequence encoding a protein compound, GNT1-GnT-III, containing a CTS domain of N-acetylglucosaminyltransferase (GNT1), fused with the catalytic domain of N-acetylglucosaminyltransferase III (GnT-III), the first nucleotide sequence is functionally associated with a first regulatory region that is active in the plant, and the second nucleotide sequence for encoding the target protein. The second nucleotide sequence is functionally linked to a second regulatory region that is active in the plant, and coexpressing the first and second nucleotide sequences to synthesize the target protein containing glikana with modified profile of N-glycosylation.

The first nucleotide sequence and the second nucleotide sequence, RAS is murenny above (method 3), can be in the plant temporarily or they can be expressed stably. Moreover, the first regulatory region is perhaps the first tissue-specific promoter, and a second regulatory region is the second tissue-specific promoter. Any of the first and second tissue-specific promoter can be a promoter of plastocyanin.

Also presented are the means (4) for the synthesis of the target protein with a modified profile of the N-glycosylation containing coexpressed within the plant, plant part or plant cell a nucleotide sequence encoding a first nucleotide sequence encoding a protein compound, GNTI-GalT containing CTS domain of N-acetylglucosaminyltransferase (GNT1), fused with the catalytic domain of the beta 1,4 galactosyltransferase (GalT), the first nucleotide sequence is functionally associated with a first regulatory region that is active in the plant, a second nucleotide sequence encoding a beta 1,4 galactosyltransferase, the second nucleotide sequence is functionally linked to a second regulatory region, which active in the plant, the third nucleotide sequence encoding a target protein, the third nucleotide sequence is functionally connected with the third regulatory region which is active in the plant, and coexpression first, second and third sequences for the synthesis of the target protein containing glikana with modified profile of N-glycosylation.

The first nucleotide sequence and the second nucleotide sequence, the above (4)may be located in the plant temporarily or they can be expressed stably. Moreover, the first regulatory region may be the first tissue-specific promoter, and a second regulatory region is the second tissue-specific promoter. Any of the first and second tissue promoter can be a promoter of plastocyanin.

In the present invention proposes a method (5) for the synthesis of the target protein with a modified profile of the N-glycosylation containing coexpressed within the plant, plant part or plant cell a nucleotide sequence that encodes a first nucleotide sequence encoding a protein compound, GNTI-GnT-III, containing a CTS domain of N-acetylglucosaminyltransferase (GNT1), fused with the catalytic domain of N-acetylglucosaminyltransferase III (GnT-III), specified the first nucleotide sequence is functionally linked to a first regulatory region that is active in the plant; the second nucleotide sequence of N-acetyl glucosaminyl transferase III, the decree is bedroom second nucleotide sequence is functionally linked to a second regulatory region, which is active in the plant; and a third nucleotide sequence encoding a target protein, specified third nucleotide sequence is functionally connected with the third regulatory region that is active in the plant; and coexpression first, second and third sequences for the synthesis of the target protein containing glikana with modified profile of N-glycosylation.

The first nucleotide sequence, the second nucleotide sequence and the third nucleotide sequence, the above (5), may be in the plant temporarily or they can be expressed stably. Moreover, the first, second and third regulatory region are possible tissue promoters. For example, any of the tissue of the promoter can be a promoter of plastocyanin.

[0030] In accordance with the described methods protein can thus be obtained in large quantities. There are no glikana, which are known to be involved in hypersensitivity reactions or, in other words, in allergic reactions. This is achieved by coexpressing glycoengineering enzymes together with the target protein, resulting in protein, which is less immunogenic than the protein obtained in the wild plant.

As discussed in n the standing description you can use the simplified expression systems to obtain the target protein using transient expression. However, these methods can be used with the use of stable conversion systems. For this reason, the present invention is not limited to using only transient expression systems.

Using the transient coexpression considered in the present description, the system avoids long periods of production, and the process of selection of elite mutants and glycoengineering transgenic lines and their subsequent use as the parent cell lines (e.g., as described in Bakker et al., 2005). The system also allows you to prevent the side effects often associated with mutants and glycoengineering plants relative to productivity, pollen productivity, tying seeds (Bakker et al., 2005), as well as the viability (Now et al., 2005). As explained in the present description, coexpressed target protein modified with chimeric human galactosyltransferase did not affect the kinetics of production and quantity of the product

The transient expression system considered in the present description, provides levels of expression, reaching 1.5 g of high quality antibody per kilogram of the assy crude tissue of leaves, that exceeds the known reports the level of accumulation of any antibody in plants using other expression systems, including systems based on many viruses and transgenic plants.

Outlined in this description of the method, for example, which includes the expression of Galt or Galt-GNT1, can also be applied to stably transformed plants. Along with the demonstration of the many advantages of the present invention is not limited to systems transient expression,

The following summary of the invention does not set forth his mandatory all distinctive features of the present invention.

BRIEF DESCRIPTION of DRAWINGS

These and other features of the present invention will become more apparent from the following description in which references to the accompanying drawings.

On figa shown based on plastocyanine cluster assembled for the expression of C5-1. R610 contains a nucleotide sequence encoding a C5-1 LC C5-1 HC-KDTL; R612 contains a nucleotide sequence encoding a C5-1 LC and C5-1 NS, where C5-1 LC: sequence encoding the light chain C5-1; C5-1 NS: sequence encoding the heavy chain C5-1. On FIGU shows the nucleotide sequence for the promoter of plastocyanin and 5'-UTR (untranslated region) (SEQ ID NO:23), the site initiated the project for the transcription shown in bold, and the initiation codon broadcast underlined. On figs shows the nucleotide sequence for plastocyanin 3'-UTR and terminator (SEQ ID NO:24), terminator underlined.

Figure 2 shows the accumulation of antibodies C5-1 in leaves of Nicotiana benthamiana, impregnated R610 and R612 (polygenic expressing clusters based on plastocyanine) with and without co-expression of suppressor HcPro silencing. Presented values correspond to the average level of accumulation and standard deviation obtained from 6 measurements at three plants (with infiltration by means of a syringe) or 6 measurements on individual batches of approximately 12 infiltrated plants (250 g).

Figure 3 shows blot analysis of protein accumulation C5-1 antibody in extracts, infiltrated using a syringe and plants with vacuum infiltration. On figa shows the immunoblotting conjugated with peroxidase antibodies goat against mouse IgG (IgG (H+L)) on extracts from plants infiltrated with the use of R612 (for secretion, track 1) or R610 (for retention of ER (endoplasmic reticulum), lane 2). C1: 100 ng commercial immunoglobulin mouse IgG1 (Sigma M9269), loaded to control the electrophoretic mobility; C2: 12 µg of total protein extracted from biomass, infiltrated the soapwort drug (empty vector. With3: 100 ng commercial immunoglobulin mouse IgG1 (Sigma M9269), added to 12 µg of total protein extracted from biomass, infiltrated the soapwort drug (empty vector). On FIGU shows the immunoblotting activity using conjugated with anti-human immunoglobulins IgG1 on extracts from plants infiltrated with the use of R612 (for secretion, track 1) or R610 (for retention of ER (endoplasmic reticulum), lane 2). C1: 2 μg control antibody C5-1, refined from hybridoma (Gaudi et al., 1997); (C2: 75 µg of total protein extracted from biomass, infiltrated the soapwort drug (empty vector).

Figure 4 shows the analysis of antibodies, refined from plants infiltrated with the use of R612 (for secretion, track 1) or R610 (for ER retention, lane 2). On figa shows the analysis by the method of polyacrylamide gel electrophoresis (SDS-PAGE) of total extracts and refined proteins performed in nevosstanovlenie conditions. On FIGU shows the analysis of SDS-PAGE of refined antibodies performed in reducing conditions. On figs shows the immunoblotting activity of refined antibodies performed using conjugated with peroxidase IgG person. On fig.4D shows the comparison of contaminants in 6 batches of refined antibody C5-1 of RA is personal infiltrated cultures. With: 2.5 mcg commercial immunoglobulin mouse IgG (Sigma M9269), loaded to control the electrophoretic mobility.

Figure 5 presents examples of polygenic clusters collected for the expression of native (R622) and hybrid (R621) versions galactosyltransferase. GNT1-CTS: CTS domain of N-acetylglucosaminyltransferase I; GalT-Cat: catalytic domain of human β1,4 galactosyltransferase; GalT (R622): human (31,4 galactosyltransferase. On FIGU shows the nucleotide sequence (SEQ ID NO: 14) for GalT (UDP GakbetaGlcNac beta 1,4 galactosyltransferase polypeptide 1, beta 1,4 galactosyltransferase I), the site of the initiation ATG is underlined; the transmembrane domain is underlined and italicized; the sequence in bold corresponds to the catalytic domain of human beta 1,4 galactosyltransferase (human beta 1,4GalT); FLAG-epitope in italics. On figs shows the amino acid sequence (SEQ ID NO: 15) for GalT (UDP-Gal:betaGlcNac beta 1,4 galactosyltransferase polypeptide 1, beta 1,4 galactosyltransferase I). The transmembrane domain is underlined and italicized; the sequence in bold corresponds to the catalytic domain of human beta 1,4 galactosyltransferase; FLAG epitope in italics. On fig.5D shows the nucleotide sequence (SEQ ID NO: 17) GNTIGalT, site initiation ATG in the will Charcot; the transmembrane domain (CTS) is underlined and italicized; the sequence in bold corresponds to the catalytic domain of human beta 1,4 galactosyltransferase (human beta 1,4GalT); FLAG epitope in italics. On file shows the amino acid sequence (SEQ ID NO: 18) GNTIGalT, transmembrane domain (CTS) is underlined and italicized; the sequence in bold corresponds to the catalytic domain of human beta 1,4 galactosyltransferase (human beta 1,4GalT); FLAG epitope in italics. On fig.5F shows the nucleotide sequence of the domain CTS (intracellular end element, a transmembrane domain, inter-zonal region) N-acetylglucosaminyltransferase (GNT1; SEQ ID NO: 21). On fig.5G amino acid sequence, aimed at the capsid (CTS) (SEQ ID NO: 22). On fign shows the sequence of the nucleic acid Gntl-Gnt III (SEQ ID NO: 26). On Fig shows the amino acid sequence Gntl-Gnt III (SEQ ID NO: 20). On fig.5J shows the sequence of the nucleic acid Gnt III (SEQ ID NO: 16). On FIGC shows the amino acid sequence of Gnt III (SEQ ID NO: 19).

Figure 6 shows the profile of the extracts obtained from expressing antibodies C5-1 plants and either stained for protein excretion, or for immunobloting. The upper panel shows the stained class "Kumasi" acridity gel (SDS page). The second top panel shows the determination of affinity of agglutinin Erythrina cristagali (ECA), which specifically binds β1,4 galactose. The third top panel shows immunoblotting using antibodies against α1,3 fucose. The bottom panel shows immunoblotting using antibodies against β1,2 xylose. R612: C5-1 expresses alone; R612+R622: C5-1 coexpression (nonfiltered) with GalT; R612+R621: C5-1 coexpression with GNT1-GalT.

7 shows MALDI-TOF mass spectrometry to identify N-glycosylation typical glycopeptide EEQFNSTFR (SEQ ID NO: 13) C5-1 antibody, isolated from plants infiltrated Nicotina benthamiana using a syringe, using a number of models. N-glycosylamine of glycopeptide was determined after separation on a preparative high performance liquid chromatograph (HPLC). Similar results were obtained after vacuum infiltration. On figa shows MALDI-TOF mass spectrometry typical glycopeptide following expression R612: (C5-1; see figure 1). On FIGU shows MALDI-TOF mass spectrometry typical glycopeptide followed by expression of C5-1 (R612; see figure 1) together with the natural GalT (R622; see figure 5). On figs shows MALDI-TOF mass spectrometry typical glycopeptide followed by expression of C5-1 (R612; see figure 1) together with GNTIGalT (R621, see figure 5). On figs - liner corresponding to the increased mean m/z 2650-280 spectrum shown (arrow) lack of comprehensive primary ion J, found in the plant R612. A: GlcNAcMan3GlcNac2; In: Man5GlcNac2; With: GalGlcNAcMan3GlcNAc2; D: GlcNAc2Man3GlcNac2; E: Man6GlcNac2;:F: GalGlcNAcMan3(Xyl)GlcNAc2; G: GlcNAcMan5GlcNac2; H: GlcNAc2Man5(Fuc)GlcNAc2; I: Man7GlcNac2; J: GlcNAc2Man3(Xyl)(Fuc)GlcNAc2; K: GalGlcNAcMan5GlcNAc2L: Man8GlcNac2; M: Man9GlcNac2.

DETAILED DESCRIPTION

The present invention relates to a method of modifying the production of glycoproteins in plants. The present invention also provides plants with modified production of glycoproteins.

In the present invention contains a description of the expression system in plants to control the expression of target protein in the plant. When using the described system, the expression of a target protein having a modified glycosylation pattern, for example, with reduced fokusirovanie, kilogramovaya or both fokusirovanie and kilogramovaya can be obtained N-glikana. In an alternative embodiment, can be derived target protein having a modified glycosylation pattern, where the protein has no fokusirovanie, xylotriose or both, and also with the holding increased Galatasaray. In addition, as stated in the description, the modulation of post-translational modifications, such as attaching the terminal galactose, reduces fokusirovanie and xylotriose expressed protein target. For example, the target protein may contain less than 10% of products fokusirovanie and xylotriose (i.e. less than 10% of residues of N-glycans fokusirovanie and xylotriose), or less than 5% products fokusirovanie and xylotriose (i.e. less than 5% of residues of N-glycans fokusirovanie and xylotriose), or less than 1% of products fokusirovanie and xylotriose (i.e. less than 1% of residues of N-glycans fokusirovanie and xylotriose) from about 0.1 to about 2% of residues of N-glycans fokusirovanie and xylotriose, from about 0.5 to about 1.5% of residues of N-glycans fokusirovanie and xylotriose, or from about 0.7 to about 1% of residues of N-glycans fokusirovanie and xylotriose, when compared with the same target protein obtained in the wild plant. Thus, the target protein can be produced in large quantities and contain no glikana, which may cause hypersensitivity reactions or can in some way be involved in allergic reactions.

[0046] non-restrictive example of the target protein to be ekspressirovali, involves a complex protein, such as antibodies is. The expression of this protein complex within agroinfiltration plants, for example Nicotina benthamiana, has reached a level of production of the protein at 1.5 g/kg wet tissue (about 25% TSP (transferred solid phase = transferred phase with immobilized antibodies). Achieved levels in 558 and 757 mg/kg/mass of raw fabric for forms of the target protein, secreted and hold ER, respectively. In the proposed non-restrictive example, the specified level of expression was achieved for antibodies, this level three times higher than the level achieved for antibodies obtained using mnogoiarusnui system transient expression (Girth et al., 2006).

[0047] the Influence of differences between the plant and the typical animals of the N-glycosylation was the main problem that caused the decision to use plants for the production of medicines. Education glycans in plants can cause a reduction in half-life grown in the plant protein in the bloodstream, or the glikana can cause patients allergic reactions.

The presence of core α1,3 fucose and β1,2 xylose in glycoproteins derived from plants, was perceived by the industry as a regulatory challenge, as they were also found in some vegetable allergens. In addition, at present documented proof is about, the destruction of the cow fucose, even α1,6 fucose, which can be found in immunoglobulins from cell SNO, will increase the activity of antibody-dependent cleocinonline cytotoxicity (ADCC). Sign considered in the description of the system is the ability to perform modulation of post-translational modifications, such as a persistent connection terminal galactose and reducing fokusirovanie and xylotriose (when compared to the same protein obtained in the wild plant). Alternatively, the level of fokusirovanie can be reduced while increasing the volume of galactosylceramide (again, when compared to the same protein obtained in the wild plant).

Changes in the volume of fokusirovanie, xylotriose or galactosylceramide can be determined by any suitable method, for example using antibodies against α1,3 fucose to detect the presence or absence of immunomagnetic about fucose (fokusirovanie), antibodies against β1,2 xylose to detect xylotriose, or the presence or absence of immunomagnetic on xylose, for example, as shown in Fig.6. In an alternative embodiment, can be applied MALDI-TOF mass spectrometry to identify N-glycosylation profile of a protein or part protein, as shown in Fig.7. Can also be used etc is another well-known specialist in this field of knowledge of the method of determination of N-glycosylation profile of a protein or part protein.

As discussed in detail below, was used system agroinfiltration based on the vacuum, which was found to be suitable for obtaining the target protein, such as antibodies from the point of view of quantity, quality and reproducibility. The transition to scalable infiltration technology allows you to receive daily grams of the specified antibodies within a small experimental setup that allows to use such a system transient expression with the purpose of obtaining materials for clinical trials within an extremely short timeframe for delivery of the licensed products on the market in quantities of up to several kilograms per year. High-quality antibodies were obtained from subject to infiltration of leaves after a single transaction affinity chromatography. However, it should be understood that the described method can also be applied to a stably transformed plants.

Posttranscriptional gene silencing (PTSG) can be applied to limit the expression of transgenes in plants, as well as the co-expression of suppressor of silencing, for example, but not limited to, HcPro from potato virus Y can be used to counter specific destruction of the transgene mRNA (Brigneti et al., 1998). Alternate the nye suppressor known from the existing prior art and can be used as follows, as set forth in the present description (Chiba et al., 2006, Virology 346; 7-14; included in the description by reference), for example, but not limited to, virus engraving tobacco TEV-p1/HC-Pro (Tobacco etch virus-p1/HC-Pro), BYV-p21, p19 of bushy dwarf virus tomato (TBSV P19), capsid protein of the virus curl of tomato (TCV-CP), cucumber mosaic virus 2b (CMV-2b), p25 of potato virus X (PVX-p25), p11 of potato virus M (PVM-p11), p11 of potato virus S (PVS-p11), P16 virus burn blueberries (BScV-p16), p23 virus Tristeza citrus (CTV-p23), p24 virus twisting grape leaves-2 (Grapevine leafroll-associated virus-2) (GLRaV-2 p24), p10 virus of grapes A (GVA-p10), p14 virus of grapes (GVB-p14), p10 latent virus of cow-parsnip (Heracleum latent vims) (HLV-p10), or P16 common latent virus garlic (Garlic common latent virus) (GCLV-pl6). Thus, the suppressor of silencing, for example HcPro, TEV-p1/HC-Pro, BYV-p21, TBSV-p19, TCV-CP, CMV-2b, PVX-p25, PVM-p11, PVS-p11, BScV-p16, CTV-p23, GLRaV-2 p24, GBV-p14, HLV-p10, GCLV-p16 or GVA-p10 can be coexpression or GalT. GNT1-GalT, GnT-III, GNT1-GnT-III, or in the above combination, to further ensure high levels of getting protein in plants.

Painting class "Kumasi" purified products obtained through the use of transient expression, indicates the presence of different rare foreign substances. These fragments, obviously, are related to the product itself, and all foreign material weighing more than 70 kDa, contained as min is minimum, one antifatigue agent (Fab-fragment), as shown by blot activity (pigv). The nature and amount related to the product of extraneous substances present in the extracts of plants, similar to that observed in systems producing mammalian cells. Therefore, the cleaning process is commonly used for treatment of certain antibodies (e.g., anion exchange, affinity, cation exchange) easily provides the required administrative bodies of the degree of purity of the protein used for therapeutic purposes.

In the present invention proposes a method of synthesizing a target protein in plants characterized by the presence of a modified glycosylation pattern. The method includes coexpression target protein with a nucleotide sequence that encodes a beta-1,4 galactosyltransferase (GalT; SEQ ID NO: 14), for example, but not limited to, GalT mammal or GalT person, however, can also be used GalT received from another source. The catalytic domain of GalT (e.g., nucleotides 370-1194 sequence SEQ ID NO: 14, bold fig.5b, or nucleotides 238-1062 sequence SEQ ID NO: 17, bold fig.5d) can also be merged with the CTS domain (i.e. cytoplasmic tail segment, a transmembrane domain, a stem region) N-acetyl shall glucosaminyl transferase (GNT1: for example, containing nucleotides 34-87 sequence SEQ ID NO: 17; fig.5d) and encodes the amino acid sequence containing amino acids 12-29 of the sequence SEQ ID NO: 18 (fige), for the formation of a hybrid enzyme GNT1-GalT, and the hybrid enzyme coexpression with the target protein. The method also includes coexpression target protein with a nucleotide sequence encoding a N-acetylglucosaminyltransferase III (GnT-III; SEQ ID NO: 16; fig.5j), for example, but not limited to, GnT-III mammal or GnT-III person, however, can also be used GnT-III, obtained from another source. In addition, the hybrid enzyme GNT1-GnT-III (SEQ ID NO: 26); fig.5d)containing cytoplasmic tail segment GNT1, merged with GnT-III, may also be used, and its description is given below.

Alternative ways of attaching a nucleotide sequence that encodes galactosyltransferase mGalT or hGalT or GalT from other sources, include the accession of GalT to the HDEL sequence, KDEL (both are sequences hold endoplasmic reticulum), cytoplasmic tail segment (CTS) protein involved in the biosynthesis of N-glycoprotein, such as, but not limited to, CTS glucosidase I, xylosyltransferase CTS glucosidase II, CTS mannosidase I, CTS beta-1,2 xylosyltransferase, CTS alpha-1,2 fucosyltransferase is. The catalytic domain of GalT may be attached to the HDEL sequence, KDEL (both are sequences hold endoplasmic reticulum) cytoplasmic end segments, i.e. CTS glucosidase I, CTS glucosidase II, CTS mannosidase I, CTS mannosidase II, CTS beta-1,2 xylosyltransferase, CTS alpha-1,2 fucosyltransferase.

The use of hybrid enzyme containing either the sequence GNT1-GalT, or sequence GNT1-GnT-III, puts the catalytic domain of GalT or GnT-III in the apparatus of the CIS-Golgi that is experiencing the early stages of a complex process of maturation of N-glycans. To test theory practice showed that blocking the activity of GalT at an early stage of maturation, the glycans can lead to accession β1,4-galactose to maturing picenum, as well as to effective inhibition of fokusirovanie and xylotriose protein, which otherwise might occur in the plant. Similarly, blocking the activity of GnT-III at an early stage of maturation, the glycans can lead to the attachment of GlcNAc residues to beta mannose for education fissile GlnAc, and lead to efficient inhibition of fokusirovanie and xylotriose protein, which otherwise might occur in the plant. For example, the target protein can be coexpression hybrid enzyme containing CTS on the men, fused with the catalytic domain of GalT, for example GNT1-GalT (R621; figa, 5d) or GNT1-GnT-III (SEQ ID NO: 26; fig.5h). If the target protein has lower levels of fokusirovanie, while required xylopyranose and galactoglucomannan proteins, together with the target protein can be expressed unmodified (native) GalT. If you want to target a protein having a modified glycosylation containing fissile remains GlnAc, together with the target protein can be expressed unmodified (native) GnT-III. For the specialist in this field of knowledge should be obvious that the optimized sequence of the nucleic acid plants can be used to obtain the unmodified or native enzyme GalT or GnT-III.

Thus, it is proposed nucleotide sequence containing the nucleotide 1-10662 sequence SEQ ED NO: 17 (GNT1-GalT) or containing the nucleotide sequence showing from 80 to 100% identity with nucleotides 1-102 sequence SEQ ID NO: 17, where the nucleotide sequence encodes a protein that modifies glycosylation of a target protein. This sequence can be optimized on the plant. Also offers a nucleotide sequence that contains the sequence of the nucleic acid having the Amu is tidy 1-1224 sequence SEQ ID NO: 14 (GalT), or containing the nucleotide sequence showing from 80 to 100% identity with nucleotides 1-1224 sequence SEQ ID NO: 14, where the nucleotide sequence encodes a protein that modifies glycosylation of a target protein. This sequence can be optimized on the plant. The sequence identity is determined using the following parameters: Program: blastn; Database: number; Expect 10; filter: low complexity; Location: in pairs; word Size: 11.

Also in the description presents the amino acid sequence shown in the sequence SEQ ID NO: 18 (GNT1-GalT; fige) or SEQ ID NO: 15 (GalT; figure 5 C).

Features nucleic acid having a nucleotide sequence containing nucleotides 1 1641 sequence SEQ ID NO: 26; (GNT1-GnT-III), or containing the nucleotide sequence showing from 80 to 100% identity with nucleotides 1-1641 sequence SEQ ID NO: 26, where the nucleotide sequence encodes a protein that modifies glycosylation of a target protein. This sequence can be optimized on the plant. Also presents the nucleotide sequence that contains the sequence of the nucleic acid having the nucleotide 232-1641 sequence SEQ ID NO: 26 (GnT-III; or nucleotides 1-1460 is posledovatelnosti SEQ ID NO: 16), or containing a sequence showing from 80 to 100% identity with nucleotides 232-1641 sequence SEQ ID NO: 26 (or nucleotides 1-1460 sequence SEQ ID NO: 16), where the nucleic acid sequence encodes a protein that modifies glycosylation of a target protein. This sequence can be optimized on the plant. The sequence identity is determined using the following parameters: Program: blastn; Database: number; Expect 10; filter: low complexity; Location: in pairs; word Size: 11 specified, the nucleotide sequence encodes a protein that modifies glycosylation of a target protein.

Also in the description presents the amino acid sequence shown in the sequence SEQ ID NO: 20 (GNT1-GnT-III; Fig) or SEQ ID NO: 19 (GnT-III; FIGC).

The term "modified glycosylation" of a protein means that the profile of the N-glican target protein containing a modified glycosylation (for example, as explained above), differs from the profile of the N-glican target protein obtained from wild plants. Modification glycosylamine may include the reduction or increase of the target protein in one or more than one glycine or divide GlnAc in half. For example, the target protein may show reduced xylotriose, low Fook is solirovanie, or simultaneously reduced xylotriose and low fokusirovanie. In an alternative embodiment, the profile of N-glican target protein can be modified so that the amount of glycosylation increases, and in some cases, the amount of xylopyranose, fokusirovanie, or both, may be reduced. In addition, can be produced divided in half GlnAc, which can lead to reduced fokusirovanie and xylotriose protein.

The terms "low xylotriose" and "low fokusirovanie" target protein means that the volume of xylotriose, fokusirovanie, or xylotriose and fokusirovanie simultaneously, and N-glycans detected in the protein of interest, 10% less than the amount of xylotriose, fokusirovanie, or xylotriose and fokusirovanie simultaneously, which are detected on the protein of interest produced in a wild plant, where the isolated target protein and which identifies cicloserina or fokusirovanie using similar method. For example, the target protein may contain less than 5% of residues N-glican subjected to fokusirovanie, xylotriose, or both, is less than 1% of residues N-glican detected in the protein of interest can be subjected fu is atilirovanie, xylotriose, or both, from about 0.1 to about 2% of residues N-glican detected in the protein of interest can be subjected to fokusirovanie, xylotriose, or both, from about 0.5 to about 1.5% of residues N-glican fokusirovanie and xylotriose, or from about 0.7 to about 1.0% residue N-glican fokusirovanie and xylotriose, when compared with the target protein obtained in the wild plant.

As shown in Fig.6 and 7, when using set forth in the description of the method can be made the target protein, showing a modified glycosylation profile. For example, received the target protein with immunological undetectable residues fucose and xylose, while the target protein coexpression with GNT1-GalT. MALDI-TOF analysis of the epitope of the target protein shows what can be derived target protein with modified glycosylation pattern at the time when the target protein coexpression or GalT or GNT1-GalT. (see Fig, 7A-C, insert). For example, on figa shows the glycosylation profile of the epitope of the target protein, expressed in the wild plant. Target protein contains several xulosalarining and fokusirovannyi residues (peaks H and J, respectively, figa). These balances are reduced or absent when the target issue for lighting the th protein coexpressed with GalT (pigv). Moreover, there are new xylopyranose residues (peak F), and the increase in balances of glycosylation in the protein of interest by co-expression with GalT (cm. peaks C, F, K, pigv). Coexpressed target protein with GNT1-GalT leads to the formation of the profile of the N-glican, characterized by the presence of less than 1% xulosalarining and fokusirovannyi residues (see figs, insert), and increased glycosylation residues (peak K, figs).

Thus, the present invention proposes a method (method a) synthesis of the target protein with modified N-glycosylation, providing the plant containing the nucleotide sequence encoding a first nucleotide sequence encoding a beta-1,4 galactosyltransferase person specified by the first nucleotide sequence is functionally linked to a regulatory region that is active in the plant, and the second nucleotide sequence for encoding the target protein, the specified second nucleotide sequence is functionally linked to a regulatory region that is active in plants, plant growth, and expression of the first and second nucleotide sequences to synthesize the target protein containing the modified N-glycosylation. Coexpressed the first sequence, the code is highlighted GalT, along with the second sequence that encodes a protein of interest, leads to the attachment of a beta-1,4 galactose to maturing picenum the target protein, thereby reducing fokusirovanie, xylotriose glycans of the target protein or both. Moreover, using this method, the degree of galactosylceramide target protein can be increased in comparison with the volume of galactosylceramide target protein obtained from wild plants, which expresses not GalT.

Also proposed a method (method B) for the synthesis of the target protein with modified N-glycosylation, which includes the expression in transient form within a plant or plant part, the nucleotide sequence that encodes a first nucleotide sequence encoding a beta 1,4 galactosyltransferase person (GalT; SEQ ID NO: 14; fig.5b)specified by the first nucleotide sequence is functionally linked to a first regulatory region that is active in the plant, and the second nucleotide sequence for encoding the target protein, the specified second nucleotide sequence is functionally linked to a second regulatory region that is active in the plant, and the expression the first and second nucleotide sequences to synthesize the target issue for lighting the th protein, containing glikana with modified N-glycosylation. Coexpressed the first sequence, the coding GalT, along with the second sequence that encodes a protein of interest, leads to the attachment of a beta-1,4 galactose to maturing picenum the target protein, thereby reducing fokusirovanie. xylotriose glycans of the target protein or both. Moreover, the degree of galactosylceramide target protein can be increased in comparison with the volume of galactosylceramide target protein obtained from wild plants, which expresses not GalT. Step expression may also include a transient coexpression the first and second nucleotide sequences or stable coexpression the first and second nucleotide sequences.

In the present invention proposes an alternative method (method C) for the synthesis of the target protein with modified N-glycosylation, providing the plant containing the nucleotide sequence encoding a first hybrid nucleotide sequence encoding a first protein compound containing CTS domain protein compound containing CTS domain. N-acetyl glucosaminyl transferase (GNT1), fused with the catalytic domain of GalT (beta 1,4 galactosyltransferase person; GNT1-GalT; SEQ ID NO: 17; fig.5d)specified first is I hybrid nucleotide sequence functionally linked to a first regulatory region, which is active in the plant, and a second nucleotide sequence encoding a target protein, the specified second nucleotide sequence is functionally linked to a second regulatory region that is active in plants, plant growth, and expression of the first hybrid and the second nucleotide sequences to synthesize the target protein containing the modified N-glycosylation. Coexpressed first hybrid sequence that encodes a GNT1-GalT, along with a second nucleotide sequence that encodes a protein of interest, leads to the attachment of a beta-1,4 galactose to maturing picenum the target protein, thereby reducing fokusirovanie. xylotriose glycans of the target protein. For example, the degree of fokusirovanie, xylotriose or both, may be reduced from about 0.5 to about 5% or from about 0.5 to about 2% by volume when compared with the target protein obtained from wild plants, which expresses not GNT1-GalT.

An additional alternative method (method D) for the synthesis of the target protein with modified N-glycosylation, which includes the expression in transient form within a plant or plant part, the nucleotide sequence that encodes the first is ibrido nucleotide sequence, encoding GNT1-GalT, the specified first hybrid nucleotide sequence functionally linked to a first regulatory region that is active in the plant, and the second nucleotide sequence for encoding the target protein, the specified second nucleotide sequence is functionally linked to a second regulatory region that is active in the plant, and the expression of the first and second nucleotide sequences to synthesize the target protein containing glikana with modified N-glycosylation. Coexpressed first hybrid sequence that encodes a GNT1-GalT, along with the second sequence that encodes a protein of interest, leads to the attachment of a beta-1,4 galactose to maturing picenum the target protein, thereby reducing fokusirovanie and xylotriose glycans of the target protein. For example, the degree of fokusirovanie, xylotriose or both may be reduced from about 0.5 to about 5%, or from about 0.5 to about 2% by volume when compared with the target protein obtained from wild plants, which expresses not GNT1-GalT. Step expression may also include a transient coexpression the first and second nucleotide sequences or stable coexpression the first and second nucleotide placenta is valnontey.

The present invention offers an additional alternative method (method E) synthesizing the target protein with modified N-glycosylation, which includes the provision of a plant containing the nucleotide sequence encoding a first nucleotide sequence encoding a beta 1,4 galactosyltransferase person (GalT), the aforementioned first nucleotide sequence is functionally linked to a regulatory region that is active in the plant, and the second nucleotide sequence for encoding the target protein, the specified second nucleotide sequence is functionally linked to a regulatory region that is active in the plant, and a third nucleotide sequence to encode a suppressor of gene silencing, for example HcPro, specified third the nucleotide sequence is functionally linked to a regulatory region that is active in plants, plant growth, and expression of the first, second and third nucleotide sequences to synthesize the target protein containing the modified N-glycosylation. Coexpressed the first sequence, the coding GalT, along with a second nucleotide sequence that encodes a protein of interest, leads to join the beta,4 galactose to maturing picenum target protein, thereby reducing fokusirovanie and xylotriose glycans of the target protein, when compared with the target protein, derived from a wild plant that does not expresses GalT. The degree of galactosylceramide target protein can be increased when compared with the target protein, derived from a wild plant that does not expresses GalT. The third expression of the sequence encoding the suppressor of silencing, provides a high output galactosyltransferase and the target protein.

In addition, we propose a method for the synthesis of the target protein (method E) with modified N-glycosylation, which includes the expression in transient form within a plant or plant part, the nucleotide sequence that encodes a first nucleotide sequence encoding a beta 1,4 galactosyltransferase person (GalT), the aforementioned first nucleotide sequence is functionally linked to a regulatory region that is active in the plant, and the second nucleotide sequence for encoding the target protein, the specified second nucleotide sequence is functionally linked to a regulatory region that is active in the plant, and a third nucleotide sequence to encode a suppressor of gene silencing, for example HcPro, specified third nucleotide of th is sequence functionally linked to a regulatory region, which is active in plants, plant growth, and expression of the first, second and third nucleotide sequences to synthesize the target protein containing the modified N-glycosylation. Coexpressed the first sequence, the coding GalT, along with a second nucleotide sequence that encodes a protein of interest, leads to the attachment of a beta-1,4 galactose to maturing picenum the target protein, thereby reducing fokusirovanie and xylotriose glycans of the target protein. The degree of galactosylceramide target protein can be increased when compared with the target protein, derived from a wild plant that does not expresses GalT. The third expression of the sequence encoding the suppressor of silencing, provides a high output galactosyltransferase and the target protein. Step expression may include a transient coexpression the first and second nucleotide sequences or stable coexpression the first and second nucleotide sequences.

In this regard, the present invention proposes a method (method E) synthesizing the target protein with modified N-glycosylation, which includes the provision of a plant containing the nucleotide sequence encoding a first nucleotide serial is lnost, encoding N-acetylglucosaminyltransferase III (GnT-III), specified the first nucleotide sequence is functionally linked to a first regulatory region that is active in the plant; and a second nucleotide sequence for encoding the target protein, the specified second nucleotide sequence is functionally linked to a regulatory region that is active in plants, plant growth, and expression of the first hybrid and the second nucleotide sequences to synthesize the target protein containing the modified N-glycosylamine. Coexpressed first sequence encoding GnT-III, along with a second nucleotide sequence that encodes a protein of interest, leads to the attachment of a beta-1,4-linked residues of N-acetylglucosamine (GlnAc) to the beta-linked-mannose (divisible by two GlnAc) on ripening picano the target protein, thereby reducing fokusirovanie, xylotriose glycans of the target protein, or both, compared with a target protein produced in the wild plant not expressing GnT-III. The sequence encoding: GnT-III (first nucleotide sequence)of the target protein (the second nucleotide sequence) or the first and second nucleotide sequences can be the ü transient downregulation. If the sequence expressroute transient, it can also be used a third nucleotide sequence to encode a suppressor of gene silencing, for example HcPro, functionally linked to a regulatory region that is active in the plant, and the first, second and third sequences are expressed on the production of the target protein containing a modified glycosylation.

The present invention also proposes an alternative method (method N) for the synthesis of the target protein with modified N-glycosylation, which includes the provision of a plant, containing the first hybrid nucleotide sequence encoding a first component of a protein encoding CTS domain. N-acetylglucosaminyltransferase (GNT1), fused with the catalytic domain of GnT-III (N-acetylglucosaminyltransferase; GNT1-GnT-III SEQ ID NO: 20), the aforementioned first hybrid nucleotide sequence functionally linked to a first regulatory region that is active in the plant; and a second nucleotide sequence for encoding the target protein, the specified second nucleotide sequence is functionally linked to a regulatory region that is active in plants, plant growth, and expression of the first hybrid and the second nucleotide pic is of egovernance on the synthesis of the target protein, containing a modified N-glycosylation. Coexpressed the first sequence, the coding GNT1-GnT-III, along with a second nucleotide sequence that encodes a protein of interest, leads to the attachment of a beta-1,4-linked residues of N-acetylglucosamine (GlnAc) to the beta-linked-mannose (divisible by two GlnAc) on ripening picano the target protein, thereby reducing fokusirovanie and xylotriose glycans of the target protein. For example, the degree of fokusirovanie, xylotriose or both may be reduced from about 0.5 to about 5% or from about 0.5 to about 2% by volume when compared with the target protein obtained from wild plants, which expresses not GNT1-GnT-III. The sequence encoding: GNT1-GnT-III (first nucleotide sequence)of the target protein (the second nucleotide sequence) or the first and the second nucleotide sequence can be transient downregulation. If the sequence expressroute transient, it can also be used a third nucleotide sequence to encode a suppressor of gene silencing, for example, HcPro, functionally linked to a regulatory region that is active in the plant, and the first, second and third sequences are expressed on the production of C is left-protein, containing a modified glycosylation.

Additional modifications to the nucleotide sequence that encodes a target protein, can be produced to ensure high product yield. For example, a second sequence encoding a target protein may also be fused with the sequence, the coding sequence, which is active in the retention of the protein within the endoplasmic reticulum (ER), for example, but not limited to, the sequence KDEL (Lys-Asp-Glu-Leu) or other sequence ER-retention, for example HDEL or KKSS.

In addition, when producing the complex of the target protein, the second nucleotide sequence used in any way - And, as discussed above, can encode more than one peptide or domain protein complex. For example, when the protein is an antibody, the second nucleotide sequence may contain two nucleotide sequences 2A and 2B, each of which encodes part of the antibody, for example, the nucleotide 2A may encode the light chain and the sequence 2B can encode a heavy chain antibody. Non-restrictive examples of such structures are presented in figure 1, where the structure of each of R216 and R610 contains two second nucleotide sequence 2A, encoding light chain antibody C5-1 (C5-1 LC), NGF is associated with national regulatory region, which is active in the plant, for example, but not limited to, promoter of plastocyanin and sequence 2 that encodes a heavy chain antibody C5-1 (C5-1 NS), which are functionally linked with a regulatory region that is active in the plant, for example, but not limited to, promoter of plastocyanin containing nucleotides 556-999 on fig.1b or SEQ ID NO: 23; US7125978 included in the present description by reference). As shown in figure 1 and with reference to the R610, KDEL sequence can be fused to the c-terminal region of one of the peptides 2A and 2B, which, for example, should not be considered restrictive, the KDEL sequence can be fused to the heavy chain of the antibody to ensure retention with ER.

Each protein is encoded by the second nucleotide sequence may be glycosylated.

Target protein thus obtained using methods a - N, can be extracted from plants. Moreover, the target protein may be partially purified or purified using standard methods, as it should be known specialist in this field of knowledge.

In cases where Express a nucleotide sequence in the plant, each of the necessary nucleotide sequences can be introduced into the plant using standard methods, transformation methods, transient transformation; two or more plants, each expressible one or more of the desired nucleotide sequence can be crossed to obtain a plant that coexpressed the desired combination of nucleotide sequences, or a combination of the above methods may be combined. For example, transient expression may be accomplished using stably transformed plants expressing sequence, encoding GalT, GNT1-GalT, GalT and GNT1-GalT, GnT-III, GNT1-GnT-III, GnT-III and GNT1-GnT-III, or a combination of them.

In this regard, in the present invention is also a method (method I) production plants, which can be used as a platform for production of the target protein with modified N-glycosylation. This method includes providing a plant that expresses one or more than one first nucleotide sequence encoding GalT, GNT1-GalT, or together GalT and GNT1-GalT, as well as the expression of one or more nucleotide sequences. To obtain the target protein any second nucleotide sequence encoding a target protein, is introduced into a plant-based platform using standard methods, uses or stable transformation or transient (unstable) transformation, and the second nucleot is DNA sequence expresses so that the received target protein contains glikana with modified N-glycosylation, or a plant expressing a first nucleotide sequence that hybridizes with a second plant expressing the second nucleotide sequence, and the thus obtained target protein contains glikana with modified N-glycosylation. The target protein can be extracted from plants and, if necessary, the target protein can be isolated and purified using standard methods.

In this regard, in the present invention is also a method (method J) producing plants that can be used as a platform for production of the target protein with modified N-glycosylation. This method includes providing a plant that expresses one or more than one first nucleotide sequence encoding GnT-III, GNT1-GnT-III, or together GnT-III and GNT1-GnT-III, as well as the expression of one or more nucleotide sequences. To obtain the target protein any second nucleotide sequence encoding a target protein, is introduced into a plant-based platform using standard methods, uses or stable transformation or transient (unstable) transformation, and the second nucleotide follower of the awn expresses so that the received target protein contains glikana with modified N-glycosylation, or a plant expressing a first nucleotide sequence that hybridizes with a second plant expressing the second nucleotide sequence, and thus, the target protein contains glikana with modified N-glycosylation. The target protein can be extracted from plants and, if necessary, the target protein can be isolated and purified using standard methods.

In this regard, in the present invention is also a method (method K) producing plants that can be used as a platform for production of the target protein with modified N-glycosylation. This method includes providing a plant that expresses one or more than one first nucleotide sequence encoding GalT, GNT1-GalT, GalT and GNT1-GalT, GnT-III, GNT1-GnT-III, GnT-III and GNT1-GnT-III, or a combination of them, as well as the expression of one or more nucleotide sequences. To obtain the target protein any second nucleotide sequence encoding a target protein, is introduced into a plant-based platform using standard methods, uses or stable transformation or transient (unstable) transformation, and the second is ucleotide sequence expresses so that the received target protein contains glikana with modified N-glycosylation, or a plant expressing a first nucleotide sequence that hybridizes with a second plant expressing the second nucleotide sequence, and thus, the target protein contains glikana with modified N-glycosylation. The target protein can be extracted from plants and, if necessary, the target protein can be isolated and purified using standard methods.

The nucleotide sequence encoding GalT, GNT1-GalT, GnT-III, GNT1-GnT-III, the target protein, or a combination of them, may be codon optimized to increase the level of expression in the plant. Under the optimized codons refers to the selection of appropriate DNA nucleotides for the synthesis of building blocks of oligonucleotides structural gene or fragment, with their subsequent enzymatic Assembly, in order to achieve the use of codons in the plant.

For optimization of expression of foreign sequences in a plant nucleotide sequence that may be wild type or synthetic sequence may be used or modified as needed, so that the corresponding protein, such as GalT, GNT1-GalT, GnT-III, GNT1-GnT-III, the target protein, or a combination of them, is produced with a higher UB is its output, what could be the level of its production, encoding a protein with unmodified nucleotide sequence. For example, and it should not be considered restrictive condition, the sequence may be a synthetic sequence that is optimized for use of codons in the plant, with approximately 80% identity with the sequence of the wild type, as determined using comparative methods, such as, but not limited to, methodology BLAST (available from GenBank, using default parameters). There is an assumption that fragments or parts of the sequence that encodes a target protein or its derivatives, which demonstrates useful biological properties, such as, but not limited to, antigenic properties, can be expressed in plant tissues.

To achieve maximum levels of expression and the production of a transgenic plant protein GalT, GNT1-GalT, GnT-III, GNT1-GnT-III, as well as the target protein, the nucleic acid sequence can be verified, and the region encoding modified to optimize for gene expression in plants using a similar procedure presents Sandra et al. (Plant Cell Reports [Reports on plant cells] 15:677-681; 1996). Table of use of codons of the genes, high expression dodol the s plants have many sources, including Murray et al (Nuc Acids Res. [Experiments. n. acid] 17:447-498; 1989). In addition, the optimization sequence can also include a reduction tandem dublicate codons, the elimination of latent splicing sites, the reduction of repeated sequences (including inverted repeats), and can be determined using, for example, Leto 1,0 (Entelechon, Germany).

In this regard, the present invention proposes a method (L) for the synthesis of the target protein with modified N-glycosylation, as set forth in one of the above methods (methods A - C), where one or more than one nucleotide sequence encoding GalT, GNT1-GalT, GnT-III, GNT1-GnT-III, the target protein or a combination of them, is optimized for expression in the plant.

Thus, the present invention relates to a plant, plant cell or seed, which contain a nucleotide sequence encoding GalT, GNT1-GalT, GnT-III, GNT1-GnT-III, the target protein or combination thereof, each of the sequences functionally linked to a regulatory region that is active in the plant. Plant, plant cell or seed may also contain a second nucleotide sequence encoding one or more than one target protein is specified by the nucleotide sequence is functionally linked to one or breeches one second regulatory region, which is active in the plant. The first nucleotide sequence, the second nucleotide sequence and the first nucleotide sequence with a second nucleotide sequence can be optimized for expression codons in the plant, plant cell or seed plants.

The term "functionally linked" means that a particular sequence interact both directly and indirectly to perform intended functions, such as mediation, in gene expression or modulation. The interaction of functionally related sequences may, for example, take place through the mediation of proteins that interact with functionally related sequences. The transcriptional regulatory region of interest sequence functionally linked, when the sequence is functionally linked to allow transcription of interest sequence, subject to the involvement of the transcriptional regulatory region in mediation or modulation.

The term "plant part" means any part, derived from plants, including the plant tissue taken from plants, such as, but not limited to, leaves, foliage, stems, roots, aboveground part together with the leaves, stalk, and the additional flowering part of the plant, cells and protoplasts obtained from a plant.

The term "plant material" means any material derived from plants. The plant material may constitute the whole plant, its tissue, cells, or any faction. In addition, the plant material may contain intracellular botanicals, plant extracellular components, liquid or solid extracts from plants or their combination. Moreover, the plant material may contain plants, plant cells, tissue, liquid extract, or a combination, of the plant's leaves, stems, fruits, roots, or combinations thereof. The plant material may contain plant or part thereof, which has not been subjected to any processing operations. However, it is also assumed that the plant material may be subjected to minimal processing, as described below, or it was a more thorough treatment, including partial or thorough cleaning with the well-known in this field of knowledge of methods, including, but not limited to, chromatography, electrophoresis, etc.

The term "minimal processing" refers to plant material, such as a plant or part thereof containing the target protein that has undergone partial purification to obtain the herbal extract homogenate, faction the plant homogenate, etc. Partial purification may include, but not be limited to, the destruction of cell structures of plants and the creation thereby of a composition containing soluble plant components and insoluble vegetable components that can be separated, for example, but not limited to, centrifugation, filtration, or both. In this regard, proteins secreted in the extracellular space of the sheet or other tissues, could be easily obtained by using vacuum or extraction in a centrifuge, or tissue can be separated under pressure by passing through the rolls, or grinding, etc. with the aim of squeezing or releasing the protein from the extracellular space. Minimal processing may also include the preparation of drugs total extracts of soluble proteins, because these drugs should have a negligible contamination of secondary plant products. In addition, minimal processing may include water extraction of soluble proteins from leaves, followed by deposition (precipitation) with any suitable salt. Other ways may include extensive maceration and extraction of the juice to make direct use of the extract.

Plant material in the form of plant matter or tissue may be taken by the patient orally. Flora is the first material may be injected into the body as part of a food additive, together with food or in capsules. The plant material or the fabric can be prepared in a concentrated form with the aim of improving or increasing a pleasant taste or presents together with other substances, ingredients or pharmaceutical fillers, if necessary.

It is assumed that the plant containing the target protein, can be assigned for administration to a patient, such as an animal or person in different ways, depending on needs and situation. For example, the target protein obtained from plants, can be selected before using it in the raw, half-purified or purified form. If the protein is to be cleaned, it can be produced either edible or inedible plants. In addition, if the protein is administered orally, plant tissue can be collected and fed directly to the patient or collected plant tissue may be dried before feeding, or allow the animal to eat the plants on the range without any prior collection. Within the scope of the present invention also takes into account the possibility of the use of the collected plant tissue as a food additive in animal feed. If plant tissue is fed animal with small or without further processing, preference is sustained fashion, to enter plant tissue was edible.

As is discussed in detail in the Examples, GalT, GNT1-GalT, as well as the target protein were injected into the plant transient way. Immunological analysis using the respective antibodies showed that a protein with MB, equal to 150 kDa, was present in the transformed cells (figure 2, 3A and 3B). In addition, GalT or GNT1-GalT was detected in extracts derived from plants expressing any design and the modified N-glycosylation of the target was observed at ekspressirovannoj GNT1-GalT in the plant (6). Thus, recombinante downregulation of GalT or GNT1-GalT is active in planta.

GalT-FLAG or GNT1-GalT-FLAG (i.e. GalT or GNT1-GalT, marked on the end of the FLAG-epitope) for carrying out immunological analysis of recombinant protein in the transformants were introduced into plants. The immunoblotting using anti-FLAG antibodies can be used to demonstrate that the corresponding protein is present in the transformed cells.

The term "analog" or "derivative" includes substitution, deletion or joining a nucleotide sequence that encodes a GalT (SEQ ID NO: 14), the catalytic domain of GalT (nucleotides 368-1198 sequence SEQ ID NO: 14; coding in bold in Fig 5b) or GNT1-GalT (nucleotides 248-1077 sequence of SEQID NO: 17; coding in bold in Fig 5d) provided that the sequence encodes a protein that modifies, or when fused with the cytoplasmic end segment (CTS) GalT, modifying the glycosylation profile of the target protein upon expression in a plant, for example the reduction of fokusirovanie, xylotriose or both, glycans of the target protein, or increased glycosylation of a target protein when compared with the profile of glycosylation of a target protein in a plant, in the absence of ectopiceski downregulation of GalT (SEQ ID NO: 14) or GNT1-GalT (SEQ ID NO: 17). For example, the protein encoded by sequence, can attach terminal galactose in the process of maturation of N-glycans. Derivatives or analogs of the nucleic acid sequences typically exhibit more than 80% identity with a sequence of nucleic acids, for example from 80 to 100% sequence identity, or 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100% the identity. The sequence identity can be determined using the algorithm BLAST (GenBank: ncbi.nlm.nih.gov/cgi-bin/BLAST/) with default settings (Program: blastn; Database: number; Expect 10; filter: low complexity; Location: in pairs; word Size: 11). Analogs or derivatives thereof also include those nucleotide sequences, is the quiet crossed in the stringent conditions of hybridization, see Maniatis et al. (Maniatis et al. in publications Molecular Cloning [molecular cloning] Guidelines for conducting work in the laboratory, Cold Spring Harbor laboratory, 1982, str-389 or Ausubel et al. (Ausubel et al. (eds), 1989, Current Protocols in Molecular Biology [the Existing protocols in molecular biology, volume 1, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, p) with any of GalT sequences (SEQ ID NO: 14), GNT1-GalT (SEQ ID NO: 18), considered in the present description, provided that the specified sequence encodes a protein that modifies glycosylation profile of the target protein, for example, reducing fokusirovanie, xylotriose or both, glycans of the target protein, or increasing the glycosylation of a target protein when compared with the profile of glycosylation of a target protein in a plant, produced in the absence of GalT (SEQ ID NO: 14) or GNT1-GalT (SEQ ID NO: 17). For example, the protein encoded by sequence, can attach terminal galactose in the process of maturation of N-glycans. An example of such stringent conditions is hybridization with a suitable probe, for example, but not limited to [gamma32P]ATP-dependent labeled probe duration 16-20 h at 65°C in 7% of the LTO, 1 mm add, 0,5M Na2HPO4pH of 7.2. Then was done washing for 30 minutes at 65°C in 5% LTOs, 1 mm etc, 40 mM Na2HPO4, pH 7.2 and washing at 65°C in % LTOs, 1 mm etc, 40 mm Na2HPO4pH of 7.2. Washed in this buffer can be repeated to reduce the background.

In a similar embodiment, the present invention includes the modification of the nucleotide sequence that encodes a GnT-III (SEQ ID NO: 16), the catalytic domain of GnT-III or GNT1-GnT-III (SEQ ID NO: 20), provided that the sequence encodes a protein that modifies, or when fused with the cytoplasmic end segment (CTS) GnT-III, modifying the glycosylation profile of the target protein upon expression in a plant, for example, divide in half GlnAc, reducing fokusirovanie, xylotriose or both, glycans of the target protein, or increase glycosylation of a target protein when compared with the profile of glycosylation of a target protein in a plant, in the absence of ectopiceski downregulation of GnT-III (SEQ ID NO: 16) or GNT1 - GnT-III (SEQ ID NO: 26). Derivatives or analogs of the nucleic acid sequences typically exhibit more than 80% identity with a sequence of nucleic acids, for example from 80 to 100% sequence identity, or 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100% the identity. The sequence identity can be determined using the algorithm BLAST (GenBank: ncbi.nlm.nih.gov/cgi-bin/BLAST/) with default settings (Program: blastn; Database: number; Expect 10; Phi is Tr: low complexity; Location: in pairs; word Size: 11). Analogs or derivatives thereof also include those nucleotide sequences that are crossed in the stringent conditions of hybridization, see Maniatis et al. (Maniatis et al. in publications Molecular Cloning [molecular cloning] Guidelines for conducting work in the laboratory, Cold Spring Harbor laboratory, 1982, str-389 or Ausubel et al. (Ausubel et al. (eds), 1989, Current Protocols in Molecular Biology [the Existing protocols in molecular biology}, vol. 1, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, p) with any of the sequences GnT-III (SEQ ID NO: 16), GNT1-GnT-III (SEQ ID NO: 26), considered in the present description, provided that the specified sequence encodes a protein that modifies glycosylation profile of the target protein, for example, reducing fokusirovanie, xylotriose or both, glycans of the target protein, or increasing the glycosylation of a target protein when compared with the profile of glycosylation of a target protein in a plant, produced in the absence of GnT-III (SEQ ID NO: 16) or GNT1-GnT-III (SEQ ID NO: 26). An example of such stringent conditions is hybridization with a suitable probe, for example, among other things [gamma32P] ATP-dependent labeled probe duration 16-20 h at 65°C in 7% of the LTO, 1 mm add, 0,5M Na2HPO4pH of 7.2. Then was done washing for 30 minutes at 65°C in 5% LTOs, 1 mm etc, 40 mm a 2HPO4, pH 7.2 and washing at 65°C in 1% LTOs, 1 mm etc, 40 mm Na2HPO4pH of 7.2. Washed in this buffer can be repeated to reduce the background.

Under "regulatory region", "regulatory element" or "promoter" refers to a portion of the nucleic acid, usually but not always located in front of the region encoding the protein of the gene, which may consist of either DNA or RNA, or DNA and RNA simultaneously. When regulatory region is active and functional interaction or functionally linked to the target gene, it can lead to the expression of a target gene. The regulatory element may be capable of mediating organ specificity or controlling evolutionary or temporary activation of the gene. "Regulatory region comprises a promoter elements, the elements of the active zone of the promoter, demonstrating the basic activity of the promoter elements that are induced in response to external stimuli, the elements that contribute to the activity of the promoter, for example, a negative regulatory elements or transcriptional gene-amplifiers. Used in the present description "regulatory region" also includes elements that are active, following the transcription, such as regulatory elements, modulating gene expression, for example, launched the nnye and transcriptional gene amplifiers, translational and transcriptional gene repressor substances, upstream activating sequences, as well as the determinants of mRNA instability. The last of these elements can be located close to the region encoding.

In the context of the present description, the term "regulatory element" or "regulatory region" generally refers to DNA sequences, more often, not always, located in front of (5'-end) of the coding sequence of a structural gene, which controls the expression of coding by providing recognition RNA polymerase and/or other factors necessary to initiate transcription at a particular site. However, it should be understood that other nucleotide sequence located within introns or 3'-end sequence, can also participate in the regulation of expression of a coding region of interest. An example of a regulatory element that recognize RNA polymerase and other transcription factors to initiate a specific site, is the element of the promoter. Most, not all, eukaryotic promoter elements contain a TATA-box, i.e. conservative sequence of nucleic acid consisting of nucleotides adenosine and thymidine, usually located on the destruction of approximately 25 pairs n is cleotides front of the site of initiation of transcription. The promoter element contains the main element of the promoter responsible for the initiation of transcription, as well as other regulatory elements (listed above), the modifying gene expression.

There are several types of regulatory regions, including adjustable evolutionary induced or constitutive. Regulatory area, adjustable evolutionary or controlling differential expression controlled gene is activated within certain organs or tissues of the body at specific intervals during the period of development of the specified organ or tissue. However, some regulatory region regulated evolutionary, may in a preferred case, to be active in certain organs or tissues at specific developmental stages. They can also be active in the regulatory regime evolutionary or also on the main level in other organs or tissues of the plant. Examples of tissue-specific regulatory regions, for example see specific regulatory region, include the promoter napina, as well as the promoter cruciferin (Rask et al., 1998, J.Plant Physiol. 152; 595-599; Bilodeau et al., 1994, Plant Cell [] 14; 125-130). Specific leaves the promoter comprises the promoter of plastocyanin (US 7125978 included in the present description by reference; SEQ ID NO: 23; Fig 1b).

Induced regulatory region I which is the area can directly or indirectly activate transcription of one or more DNA sequences or genes in response to the inductor. In the absence of inducer sequence reads DNA or genes will not occur. Usually factor protein, acceding specifically to inducible regulatory region to activate transcription, may be present in an inactive form, which may be directly or indirectly converted inductor in an active form. However, the factor protein may also be absent. The inducer can be a chemical substance such as a protein, metabolite, growth regulator, herbicide, or phenolic compounds, or physiological stress caused directly by heat, cold, salt or toxic elements, or indirectly, through the effect of pathogenic or disease-causing substance, such as a virus. A plant cell containing induced regulatory region may be affected by the inductor when the external application of the inducer to the cell or plant, for example, by spraying, moisture, heat or other means. Inducible regulatory elements may be derived from genes of plant and nereshitelno origin (for example, Gatz S. Lenk, I.R.P., 1998, Trends Plant Cell. [Selection of plants, with changes] 3, 352-358 included in this description is of by reference). Examples of potential induced promoters include an example, but not limited to, induced by the tetracycline promoter (Gatz S., 1997, Ann. Rev. Plant Physiol. Plant Mol. Biol. 48, 89-108; included in the present description by reference)induced by steroid promoter (Aoyama, T. and Chua, N.H., 1997, Plant J. 2, 397-404; included in the present description by reference) and induced by ethanol promoter (Salter, M.G., et al., 1998, Plant Journal 16, 127-132; Caddick, M.X., et al., 1998, Nature Biotech. 16, 177-180, included in the present description by reference), induced by cytokinin genes IB6 and CKI1 (Brandstatter, I., and Kieber J.J., 1998, Plant Cell 10, 1009-1019; Kakimoto, T., 1996, Science 274, 982-985; included in the present description by reference), and, finally, induced by auxin element, DR5 (Ulmasov, T., et al., 1997, Plant Cell 19, 1963-1971; included in the present description by reference).

Constitutive regulatory region directs expression of genes through different parts of the plant and continuously throughout plant development. Examples of constitutive regulatory elements include promoters that are associated with the transcript of the cauliflower mosaic virus CaMV 35S (Odell et al., 1985, Nature, 313; 810-812), the rice actin 1 (Zhang et al., 1991, Plant Cell 3; 1155-1165), actin 2 (An et al., 1996, Plant J. 10: 107-121), or trimethylsilyl 2 (TMS 2) (U.S. 5,428,147 included in the present description by reference), and genes triosephosphate 1 (Xu et al., 1994, Plant Physiol. 106; 469-467), the gene of the maize ubiquitin 1 (Crmejo et al., 1993, Plant Mol. Biol. 29; 637-646), genes of the ubiquitin 1 and 6 of Arabidopsis (Holtorf at al., 1995, Plant Mol. Biol. 29; 637-646), gene factor 4A initiating broadcast of tobacco (Mandel at al., 1995, Plant Mol. Biol. 29; 995-1004). The term "constitutive"as used in the present description, does not necessarily mean that controlled constitutive gene regulatory region is expressed at the same level in all cell types, but that the gene is expressed in a wide range of cell types, despite the fact that there is often a change in the number.

One or more than one nucleotide sequence according to the present invention can be expressed in any suitable plant host, which is transformed using the specified nucleotide sequence, or structures or vectors of the present invention. Examples of appropriate hosts include, but are not limited to, agricultural crops, including alfalfa, canola, wild mustard, maize, tobacco, alfalfa (Lucerne field), potato, ginseng, pea, oat, rice, soybean, wheat, barley, sunflower and cotton.

One or more chimerical genetic structures in accordance with the present invention can further comprise a 3'-noncoding region. Specified 3'-noncoding region refers to the portion of the gene containing the DNA segment, which has the signal Palade is helirovanie and any other regulatory signals, capable of mRNA processing or gene expression. The polyadenylation signal is usually characterized by the execution of the merger traces polyadenylic acid to the 3'end of the precursor mRNA. The polyadenylation signals are usually detected by the presence of homology to the canonical form 5' AATAAA-3', although variations are not common. One or more chimerical genetic constructs of the present invention may also include additional enhancer (amplifiers), amplifiers translation or transcription, as may be required. These strengthening well-known to specialists in this field of knowledge, and they may include the initiation codon ATG and the surrounding sequence. The initiation codon must be matched in phase with the reading frame coding sequence to ensure translation of the entire sequence.

Non-restrictive examples of suitable 3' regions are the 3' transcribed non-translated regions containing the polyadenylation signal of Agrobacterium tumor Agrobacterium inducing (Ti) plasmid genes, such as Napolean synthase (gene NO-synthase)genes of a plant, such as genes protein storage of soybeans and small subunit gene of ribulose-1,5-phosphate carboxylase (ssRUBISCO).

To facilitate identification of transformed plant cells design is tion of the present invention may be subjected to additional pressure for the introduction of breeding plant markers. Useful selective markers include enzymes, which provide resistance to chemicals such as antibiotics, such as gentamicin, hygromycin, kanamycin, or herbicides, for example, phosphinotricin, glyphosate, chlorsulfuron, etc. in a Similar way can be used enzymes providing for production of the connection, determined by color change, for example, beta-glucuronidase (GUS), or luminescence, such as luciferase or green fluorescent protein (GFP).

You should also take into account the portion of the invention relating to transgenic plants, plant cells and seeds containing the construction of a chimeric gene in accordance with the present invention. Methods of regeneration of whole plants from plant cells is also known from the existing art. In General, the transformed plant cells are cultured in an appropriate environment, which may contain selective agents, such as antibiotics, which are selective markers for easy identification of transformed plant cells. After the formation of callus formation processes may be caused by the use of appropriate plant hormones in accordance with known methods, and processes are put in carpobrotus environment for plant regeneration. P is obtained plants can be used to create re-generations or from seeds or by using methods of vegetative propagation. Transgenic plants can also be grown without the use of tissue cultures.

Regulatory elements in accordance with the present invention can also be combined with interest the region coding for the expression within organisms hosts, which are amenable to transformation or transient expression. Such organisms include, but are not limited to, plants of both monocots and dicots, such as, but not limited to, grains, cereals, wheat, barley, oats, tobacco, mustard, soybean, beans, peas, alfalfa, potato, tomato, ginseng and Arabidopsis.

How stable transformation and regeneration of these organisms exist in the field of technology and well-known specialist in this field of knowledge. A method of obtaining a transformed and regenerated plants is not essential for the present invention.

The term "transformation" means the interspecies transfer of genetic information (nucleotide sequence), which is manifested genotypic, phenotypic or both type at the same time. Interspecific transfer of genetic information from chimeric constructs to the owner may be hereditary, and transfer of genetic information to be considered stable, Il the transfer may be transient, and transfer of genetic information is not to be inherited.

The present invention also includes a suitable vector containing a chimeric construction, easy to use with the system is either stable or transient expression. Genetic information can also be presented in one design and more. For example, the nucleotide sequence encoding the target protein can be introduced into a single structure, and a second nucleotide sequence encoding a protein that modifies glycosylation of a target protein, can be entered using a separate design. These nucleotide sequences can then be coexpression inside plants. But it can also be used to construct containing the nucleotide sequence encoding both the target protein and protein-modifying the glycosylation profile of the target protein. In this case, the nucleotide sequence would contain the first sequence comprising a first nucleic acid sequence encoding a target protein, and functionally related to the promoter or regulatory region, and a second sequence containing the second nucleic acid sequence encoding a protein that modifies the profile glycosyl the regulation of the target protein, the specified sequence functionally linked to a promoter or regulatory region.

The term "coexpression" implies that the two nucleotide sequences, and more Express almost simultaneously within the plant and within the same plant tissues. However, it is not necessary that the nucleotide sequence is expressed exactly in the same time. It is preferable that the two nucleotide sequences, and more expressed so that the encoded product had the opportunity to interact. For example, a protein that modifies glycosylation of a target protein, can be expressed before or during the period of expression of a target protein so that is a modification of the glycosylation of a target protein. Two nucleotide sequences, and more can be coexpression using a transient expression system in which the two sequences and more are introduced into the plant at almost the same time in the conditions under which the expression of both sequences. Alternatively, plant, platform, containing one of the nucleotide sequences, for example, a sequence encoding a protein that modifies glycosylation profile of the target protein, can be transformed either the transient or Abilene with additional sequence, encoding the target protein. In this case, the sequence encoding a protein that modifies glycosylation of a target protein can be expressed in the desired tissues at the desired stage of development, or its expression can be induced by using inducible promoter, and an additional sequence encoding a target protein can be expressed in similar conditions and in the same fabric to ensure the co-expression of nucleotide sequences.

Design in accordance with the present invention can be introduced into the plant cells using Ti plasmids, Ri plasmids, vectors of plant viruses, direct DNA transformation, microinjection, electroporation, etc. To familiarize yourself with these methods, see, for example, Weissbach and Weissbach, Methods for Plant Molecular Biology, Academy Press, New York VIII, pp.421-463 (1988); Geierson and Corey, Plant Molecular Biology, 2d Ed. (1988); and Miki and lyer, Fundamentals of Gene Transfer in Plants. In Plant Metabolism, 2d Ed. DT. Dennis, DH Turpinm DD Leferbve, Layzell DB (eds), Addison Wesly, Langmans Ltd London, pp.561-579 (1997). Other methods include direct uptake of DNA, the use of liposomes, electroporation, for example, the use of protoplasts, microinjection, micronarrative and vacuum infiltration. See, for example, Bilang et al. (Gene 100: 247-250 (1991), Scheid et al. (Mol. Gen. Genet. 228: 104-112, 1991), Guerche et al. (Plant Science 52: 111-116, 1987), Neuhause et al. (Theor. Appl Genet. 75: 30-36, 1987), Klein et al., Nature 327: 70-73 (1987) Howell et al. Science 208: 1265, 1980), Horsch et al. (Science 227: 1229-1231, 1985), De Block et al., Plant discrimination 91: 693-701, 1989)), Methods for Plant Molecular Biology (Weissbach and Weissbach, Academy Press Inc., (1988), Methods for Plant Molecular Biology (Schuler and Zielinski, eds, Academic Press Inc., 1989), Liu and Lomonossoff (J. Virol Meth, 105: 343-348, 2002), U.S. patent No. 4945050; 5036006 and 5100792, patent application U.S. reg. No. 08/438666, filed may 10, 1995 and 07/951715, filed September 25 (all included in the description by reference).

As shown in the description below, the methods of transient expression can be used for expression of the constructs in accordance with the present invention (see Liu and Lomonossoff, 2002, Journal of Virological Methods 105: 343-348); included in the present description by reference). Alternatively can be used methods of transient expression, based on the vacuum as outlined by Kapila et al., 1997, is hereby incorporated into this description by reference). These methods may include, for example, but not limited to, the method of the desired molecule or agroinfiltration, however, as noted above, can also be applied to other transient methods. By using the desired molecule or agroinfiltration mixture of agrobacteria (Agrobacteria)containing the desired nucleic acid, penetrates into the intercellular spaces of tissues, such as leaves, aerial parts of plants (including stem, leaves and flower), other parts of the plant (stem, root, flower) or the whole plant. After the recovery is of epidermis agrobacteria infect and transmit copies of T-DNA into the cells. T-DNA episomal transcribed, and the mRNA is translated, causing the production of the target protein in the infected cells, infiltration of T-DNA into the nucleus is a temporary (transient).

The terms "desired gene", "target nucleotide sequence"or "target coding region" refers to any gene that any nucleotide sequence, or the coding region, which must be expressed in the body-master, for example in the plant. These terms are used interchangeably. Such a target nucleotide sequence includes, but is not limited to, a gene or coding region, the product of which is the target protein. Examples of the target protein include, for example, but not limited to, industrial enzyme, protein Supplement, nutraceuticals, product value-added or fragments thereof for use in feed, food or both at the same time, pharmaceutically active protein, for example, but not limited to, growth factors, growth regulators, antibodies, antigens and their fragments or derivatives thereof suitable for immunization or vaccination and the like, Additional target protein can include, but are not limited to, interleukins, such as one or more IL - IL, IL-26 and IL-27, cytokines, erythropoietin (EPO), insulin, factors G-CSF, GMCSF, GGC-CSF, M-CSF, and is, and combinations thereof, interferons such as alpha interferon, beta interferon, gamma interferon, blood coagulability, for example, factor VIII, factor IX, or tap (tissue activator of plasminogen), HGH (human growth hormone), receptors, agonists of the receptors, antibodies, neuro-polypeptides, insulin, neuro-peptides, vaccines, growth factors, such as, but not limited to, keratinocyte growth factor and transforming growth factor, growth regulators, antigens, autoantigens, fragments thereof or combinations thereof.

If the target nucleotide sequence encodes a product which is directly or indirectly toxic to the plant, by using a method in accordance with the present invention such toxicity may be reduced in the entire plant by selective expression of a target gene within the desired tissue or at the desired stage of plant development. In addition, for a limited period of expression, due to the transient expression system, can reduce the effect in the production of toxic product in the plant.

Target the coding region or the target nucleotide sequence can be expressed in any plant host, which is either transformed or consists of a nucleotide sequence or nucleic acid molecules or genetically the design, or vectors in accordance with the present invention. Examples of appropriate hosts include, but are not limited to, Arabidopsis, agricultural crops, including, for example, canola (a form of rape), a kind of cabbage, corn, tobacco, alfalfa, potato, ginseng, pea, oat, rice, soybean, wheat, barley, sunflower and cotton.

As detailed in the examples below, the synthesis of the target protein, for example but not limited to antibody, C5-1, with modified N-glycosylation, was obtained in the plant coexpression or GalT or GNT1-GalT.

Sequence listing

Follower
ness
SEQ ID NOSequenceSEQ ID NO
(Identification number sequence)(Identification number sequence)
Xmal-pPlas.c1GNT1-GalT (nucleotide sequence)17

Sacl-ATG-pPlas.r2 GNT1-GalT amino acid18
Sacl-PlasTer.c3GnT-III amino acid19
EcoRI-PlasTer.r4GNT1-GnT-III amino acid20
Plasto-443c5CTS domain GNT1 (nucleotide)21
Plas+LC-C51.r6CTS domain GNT1 (amino acid)22
LC-C51.C7The promoter of plastocyanin and 5'UTR23
LC-C51XhosSac.r8Plastocyanin 3'UTR and the terminator24
Plas+HC-C51.r9FgalTSpe25
HC-C51.C10GNT1-GnT-III (nucleotide)26
HC-C51XhosSac.r11FgalT27
HC-C51KDEL(Sacl).r12RgalTFlagStu28
Trypticase glikopeptid13FGNT29
GalT I (nucleotide)14RGBTSpe30
GalTI 15

amino acid GnT-III (nucleotide)16

Examples

Example 1. Assembly polygenic expressing clusters R610, R612 (Figa), R621 and R622 (Figa)

All manipulations were carried out using common protocols in molecular biology by Sambrook and Russell (2001).

R610, R612 (Figa)

Applied oligonucleotides (primers) are as follows:

The first step cloning consisted of the Assembly of the receptor plasmid containing the higher and lower regulatory elements of the gene of plastocyanin alfalfa. The promoter of plastocyanin (U.S. Patent 7125978 included in this is e description in the form of links) and the sequence 5'-utrs were amplified from alfalfa genomic DNA using oligonucleotide primers Xmal-pPlas.c (SEQ ID NO: 1) and Sacl-ATG-pPlas.r (SEQ ID NO: 2). The resulting amplification product was processed using Xmal and Sacl and then sewn in pCAMBLA.2300 previously treated with these same enzymes, to generate pCAMBIA-PromoPlasto. Similarly the sequence of the 3'UTR and terminator gene of plastocyanin (figs; nucleotides 1-399 sequence SEQ ID NO: 24) were amplified from alfalfa genomic DNA using the following primers: Sacl-PlasTer.c (SEQ ID NO: 3) and EcoRI-PlasTer.r (SEQ ID NO: 4), and the product was processed using Sad and EcoRI before entering into the same sites of pCAMBIA-PromoPlasta to create pCAMBIAPlasto.

Plasmids R610, R612 were prepared so as to contain sequences encoding light chain antibody C5-1 and a heavy chain antibody C5-1 under the promoter of plastocyanin alfalfa as a tandem structure; R610 was created for retention in the ER gathered IHH and contained the sequence KDEL, combined with the end of the heavy chain antibody C5-1, and R612 was created to ensure secretion.

Build polygons C5-1 expressing clusters was performed using the method of PCR-mediated leading (stitching), described Darveau et al. (1995). For Assembly sequences encoding light chain, below the promoter of plastocyanin, the first step was to strengthen the first 443 base pairs (bp) of the promoter of plastocyanin alfalfa (nucleotides 556-999 Fig 1b or SEQ ID NO: 23), is isano D Aoust et al. (U.S. patent 7125978 included in the present description by reference)below in relation to the initial ATG, by PCR using pCAMBIAPlasto as template and the following primers:

Plasto s (SEQ ID NO: 5) and Plas+LC-C51.r (SEQ ID NO: 6; overlapping pointed out above).

In parallel, the sequence encoding a light chain was PCR-amplified from Plasmid pGA643-kappa (Khoudi et al., 1999) using the following primers:

LC-C51.C (SEQ ID NO: 7) and LC-C51XhoSac.r. (SEQ ID NO: 8; overlapping pointed out above).

The resulting amplification of two products were mixed and used as template in a third PCR reaction using primers Plasto-443c (SEQ ID NO: 5) and LC-C51XhoSac.r. (SEQ ID NO: 8). The overlap between primers Plas+LC-C51.r (SEQ ID NO: 6) and LC-C51.C (SEQ ID NO: 7), used in the first reaction, led to the Assembly of amplification products during the third reaction. The collected product obtained in the third PCR reaction was processed using DraIII and SacI and put into the website pCAMBIAPlasto processed using DraIII and Sad, to form plasmid R540/

Sequence encoding a heavy chain was fused with the top regulatory element of plastocyanin by amplificatore 443 base pairs above the initial ATG of plastocyanin, nucleotides 556-999 (fig.1b; SEQ ID NO: 23) using PCR with the use of the site pCAMBIAPlasto as matrix and following the law the measures:

Plasto 443 (SEQ ID NO: 5) and NS-CG (SEQ ID NO: 9; overlapping pointed out above).

The products of these reactions were mixed and collected in a third PCR reaction using primers Plasto 443 (SEQ ID NO: 5) and HC-C51XhoSac.r (SEQ TH NO: 11). The resulting fragment was treated with the use of DraIII and Sad and put in pCAMBIAPlasto between sites DraIII and SacI. The resulting plasmid was named R541.

KDEL-marker was attached to the C-end of the sequence that encodes a heavy chain by PCR amplification using primers Plasto 443 (SEQ ID NO: 8) and NA-s KDEL (Sacl).r (SEQ ID NO: 12), using plasmid R541 as a matrix. The resulting fragment was treated with the use of DraIII and SacI, and cloned into the same sites pCAMBIAPlasto, creating plasmid R550.

Build polygons expressing clusters of light and heavy chains of similar binary plasmids was performed as follows: R541 and R550 were processed using EcoR1, dephosphorylated ("blunt ends")treated with the restriction enzyme HindIII and legirovanyh in HindIII and Smal receptor zones plasmids R540 to create R610 (KDEL) and R612 (without KDEL, see figure 1).

R610, R612 (Figa) - Applied oligonucleotides (primers) are as follows:

Plasmids for the expression of GalT and GNTIGalT were collected from pBLTI121 (Pagny et al., 2003). Human gene β(,4)-galactosyltransferase (hGalT) (UDF-galactose; β-N-acetylglucosamine; β(1,4)-galactosyltransferase; EC 2.4.1.22) was isolated from pUC19-hGalT (Watzele et al.. 1991) processing EcoRI. After processing by Lenovo 1,2-celibately fragment GalT was cloned in pBLTI221 on the site Sma I, this resulted in plasmid pBLTI221 GalT. The flag is a marker was attached to the C-terminal region coding region using PCR using primers FGalT (SEQ ID NO: 27) and RGalTFlagStu (SEQ ID NO: 28) for amplification. Then was produced plasmid R622 by cloning this XbaI-StuI fragment into the binary vector pBLTI121. The first AA 77. from N-acetyl glucosaminyl transferase I (GNTI), corresponding to the transmembrane domain were PCR amplified using tobacco cDNA that encodes a N-GNTI as matrix (Strasser et al., 1999), as well as FGNT (SEQ ID NO: 29) and RGNTSpe (SEQ ID NO: 30) as primers. Product amplificatoare was first cloned in the vector pGEM-T a obtained plasmid was processed using ApaI and BamHI, and is associated with pBLTI221, producing plasmid, referred to as pBLTI221-GNTI. The catalytic domain hGalT was obtained by PCR amplification on pBLTI221 hGalT, using primers FGalTSpe (SEQ ID NO: 25) and RGalTFlagStu (SEQ ID NO: 28), with the formation of sites SpeI and StuI at the 5'-end 3'-end, respectively. Then SpeI fragment/StuI hGalT was cloned in pBLTI221-GNTI using the same (SpeI and StuI sites with the creation of pBLTI221-GNTIGalT. Finally, the site pBLTI221-GNTIGalT was processed using XbaI Is StuI with the Department GNTIGalT coding sequence (fig.5d; SEQ ID NO: 17), a R621 was obtained by cloning the specified fragment into the binary vector pBLTI121.

All clones were arranged in sequence in order to confirm the integrity of the structures. The plasmids were used to transform opuholeobraznye bacteria (Agrobacterium tumefaciens) (AGL 1; ATCC, Manassas, VA 20108, USA) by electroporation (Hofgen and Willmitzer, 1988) using the apparatus Gene Pulser II (Biorad, Hercules, CA. USA) for transformation of Escherichia coli (W.S. Dower, Electroporation of bacteria, in "Genetic Engineering", Volume 12, Plenum Press, New York, 1990, J.K. Setlow, eds.). The integrity of all strains of A. tumefaciens was confirmed by restriction mapping.

Design HcPro was prepared in accordance with the description in Hamilton et al. (2002).

Example 2. Preparation of plant biomass inoculum, agroinfiltration, the product collection

Tobacco plants were grown from seeds in a flat pallet, filled with a commercially available substrate of peat moss. Plants were grown in the greenhouse at length the period of 16/8 and temperature: 25°C day/ 20°C night. After three weeks after seeding individual shoots were collected transplanted in pots and left to grow in the greenhouse for another three weeks under the same environmental conditions.

Strains of agrobacteria R612, R610, R621, R622 or 35S HcPro were grown in YEB medium supplemented with 10 mm 2-[N-morpholino]econsultancy (MES) acid, 20 μm acetoin is she, 50 µg/ml kanamycin and 25 μg/ml of carbenicillin, a pH of 5.6, until, when they reach a density of cells OD600in the range from 0.6 to 1.6. Before using suspended agrobacteria been centrifugating, and then were resuspendable environment infiltrate (10 mm MgCl2and 10 mm MES, pH 5,6).

Infiltration using the syringe was performed as described by Liu and Lomonossoff (2002, Journal of Virological Methods 105: 343-348)

For infiltration under vacuum suspension of bacteria A, tumefaciens were zentrifugenbau were resuspendable environment infiltrate and placed in storage at night at 4°C. on the day of the infiltration samples of the cultures were diluted to 2.5 volumes of culture, and then heated before use. All tobacco plants were placed for 2 minutes in an inverted position (upside roots) in the suspended bacteria in a sealed stainless steel tanks under vacuum 20-40 Torr. After complete infiltration by means of a syringe or a vacuum plants were returned to the greenhouse for a period of incubation for 4-5 days before harvest.

The selection of leaves and total protein extraction

After incubation of the aerial parts of plants were collected, frozen at a temperature of -80°C, crushed to pieces, and divided into parts by mass of from 1.5 to 7.5 grams. Total soluble protein was extracted by the method homogenizes and (Polytron) each part of the frozen and crushed plant material in three volumes of cold 50 mm Tris solution, pH 7.4, 0.15 M NaCl, 0.1% Triton X-100, 1 mm phenylmethanesulfonyl and 10 μm of hemostatis. After gomogenizirovannykh the slurry was subjected to centrifugating at the level of 20,000 rpm for 20 minutes at 4°C, purified total extract (supernatant) was saved for analysis. The total protein content of the purified total extract was determined using analysis of Bradford (Bio-Red, Hercules, CA) using bovine serum albumin as a standard sample.

Example 3. Analysis of protein, immunoblotting and ELISA (enzyme-linked immunosorbent assay)

C5-1 is antiimmunoglobulin human/mouse, so its detection and quantitative analysis can be performed by using either its affinity (affinity) with the IHH person (blot analysis activity), or its immunoreactivity relative to antiimmunoglobulin mouse.

Protein from the full total extracts or purified antibody were separated using SDS-PAGE and either stained class "Kumasi blue R-250 and G-250 or electroparadise on the membranes of polyphenylen-fluoride (Roche Diagnostics Corporation, Indianopolos, IN) for immunodetection. Before immunoblotting membranes were blocked with 5% skimmed milk and 0.1% Tween-20 in Tris-buffered salts solution (TBS-T) for 16-18 h at 4°C.

The immunoblotting was performed by incubation with the following antibodies: conjugated with peroxidation K03S against IHH mouse (H+L) (Jackson ImmunoResearch, West Grove, PA, Cat# 115-035-146) (0.04 µg/ml in 2% skimmed milk in a solution of TBS-T), conjugated to peroxidase antibody IHH from biomaterial person (Gumunex ® Bayer Corp., Elkart, IN) (0.2 ág/ml in 2% skimmed milk in a solution of TBS-T) or polyclonal goat antibody against mouse immunoglobulin (specific for the heavy chain) (Sigma-Aldrich, St-Louis, MO) (0.25 microgram/ml in 2% skimmed milk in a solution of TBS-T). Conjugated with peroxidase antibody of the biomaterial ass against immunoglobulin G from biomaterial goat (Jackson ImmunoResearch) (0.04 µg/ml in 2% skimmed milk in a solution of TBS-T) was used as secondary antibody for membranes treated with antibody specific for the heavy chain. Immunoreactive complexes were detected by chemiluminescence using lyuminola as substrate (Roche Diagnostics Corporation). Conjugation with peroxidase-enzyme horseradish antibodies IHH from biomaterial man was made using a set of conjugation with the EZ-Link Plus ® - activated peroxidase (Pierce, Rockford, IL).

Quantitative analysis of ELISA (enzyme-linked immunosorbent assay)

Advance tablets (Immunol 2HB, ThermoLab System, Franklin, MA) were coated with 2.5 µg/ml antibody goat against IHH-specific mouse heavy chain IHH (Sigma M8770)in 50 mm carbonate buffer (pH 9,0) at 4°C for 16-18 hours and Then advance the tablets were blocked by one-hour incubation in 1% casein is in saline phosphate buffer (PBS) (Pierce Biotechnology, Rockford, II) at 37°C. was constructed calibration curve control titration of solutions of purified IgG mouse (Sigma M9269). When performing immunoassays dilution (control and sample) was carried out in the plant extract obtained from a plant tissue, infiltrated and incubated with false inoculum to exclude any influence of the matrix. The plate was incubated with protein samples by performing a calibration curve titration for one hour at 37°C. After three washes with 0.1% Tween-20 in PBS solution (PBS-T) tablets were incubated with conjugated with peroxidase antibody goat against mouse immunoglobulin G (H+L) (Jackson ImmunoResearch, 115-035-146) (0.04 µg/ml) for one hour at 37°C. Washing with PBS solution were repeated, and the tablets were incubated with the substrate 3,3'. 5,5'-tetramethylbenzidine (TMB) Sure Blue peroxidase (KPL, Gaithersburg, MD). The reaction was stopped by addition of IN HC1, and the absorption was read at 450 nm. Each sample was analyzed three times, and concentrations were interpolated on a straight-line plot of the calibration curve.

Example 4. Cleaning IHH

Cleaning C5-1 from the remnants of leaves involved the selection of frozen leaf tobacco and N. benthamiana (100-150 g), adding 20 mm sodium phosphate, 150 mm NaCl, and 2 mm sodium pyrosulfite at pH of 5.8-6.0, as well as their mixing using a commercially available blender is for 2-3 minutes at room temperature. Insoluble fiber was removed by coarse filtration material Miracloth (Calbiochem, San Diego, CA), and then to the filtrate was added 10 mm phenylmethanesulfonyl fluoride (PMSF). The extract was brought to pH values of 4.8±0,1 addition of 1 M HCL and clarified by centrifugalism with the speed of 18000 rpm for 15 minutes at 2-8°C. the Clarified supernatant was brought to pH 8.0±0.1 is added 2 M solution of TRIS (hydroxyethylaminomethyl), again clarified by centrifugalism with the speed of 18000 rpm for 15 minutes at 2-8°C, and then filtered to consistently set the membranes of 0.8 and 0.2 μm (Pall Corporation, Canada). The filtered material was subjected to concentration flowing through the filter in the flow direction using ultrafiltration membrane having an effective area of 0.2 square feet, and catching particles with a molecular weight of 100 kDa (GE Healthcare Biosciences, Canada) in order to reduce the volume of the clarified material is 5 to 10 times. Concentrated samples were applied to columns with dimensions of 5 mm x 5 cm and a volume of 1 ml, with fast flow sepharose G protein (Sigma-Aldrich, St Louis, MO, Cat. # R). The column was washed 5 times at full volume with a solution of 20 mm TRIS-HC1, 150 mm NaCl with a pH of 7.5. The antibody was extracted using 100 mm Glycine pH of 2.9-3.0 and immediately brought to neutral pH by collecting into tubes containing estimated volumes Rast is ora 1 M TRIS-HCl with pH 7.5. Combined fractions antibodies were subjected to zentrifugenbau speed 21000 rpm for 15 minutes at 2-8°C and stored at -80°C until analysis. Column for affinity chromatography was cleaned and stored in accordance with manufacturer's instructions. Such chromatographic medium can be used for multiple cleaning operations without significant changes in the characteristics of treatment (tested up to 10 cycles).

Example 5. Analysis of N-glycosylation

Samples containing antibody C5-1 (50 μg)were subjected to analysis by the method of polyacrylamide gel electrophoresis (SDS-PAGE). Heavy and light chains were detected by the method of dyeing class "Kumasi blue", and the region of localization of the protein, corresponding to the heavy chain was removed and divided into small fragments. The fragments were washed three times with 600 μl of a solution of 0.1 M NH4HCO3/CH3CN each time for 15 minutes, followed by drying.

Reduction of disulfide bonds occurred by incubation residues gel in 600 μl of a solution of 0.1 M DDT in 0.1 M NH4HCO3at 56°C for 45 minutes. Alkylation was carried out by adding 600 μl of a solution of iodoacetamide 55 mm in 0.1 M NH4HCO3at room temperature for 30 minutes. Supernatant were removed and polyamide residues were washed away should represent the time in a solution of NH 4HCO30.1 M/CH3CN (1/1).

Then proteins were processed using the 7.5 μg of trypsin (Promega) in 600 μl of 0.05 M NH4HCO3at 37°C for 16 hours. Were added 200 μl of CH3CN, and was collected supernatant. After that, the remnants of the gel were washed using 200 ál of 0.1 M NH4HCO3/CH3CN, then using 200 ál of CH3CN again, and finally, using 5% formic acid. Supernatant were combined and freeze-dried

Separation of peptides by HPLC was performed on a column with reversed phase 18 (4,5x250 mm) with a linear gradient of CH3CN in 0.1% of TN. Fractions were collected and freeze-dried, and then analyzed using MALDI-TOF mass spectrometry instrument Voyager DE-Pro MALDI-TOF (Applied Biosystems, USA)equipped with a 337-mm nitrogen laser. Mass spectrometry was performed delayed extraction reflector using as matrix alpha-cyano-4-hydroxylamino acid (Sigma-Aldrich).

Example 6. Quantitative analysis of transient expression of IHH in agroinfiltration leaves of tobacco Nicotiana benthaniana

To test the ability of the solid, based on plastocyanine polygenic expressing clusters to manage a large accumulation of the fully assembled IHH coding sequences of the light and heavy chain antibody C5-1 mouse IgG PR is against IHH person (Khoudi et al., 1997) were collected in tandem design, located below the promoter of plastocyanin and 5' netrenirovannykh sequences and bounded on the sides 3' netransliruemymi and transcription termination sequences on the same segment of T-DNA binary plasmid pCambia, as shown in figure 1.

In both polygenic expressing clusters R612 and R610 (see Example 1) sequences encoding the light and heavy chains contain native signal peptides from C5-1 (Khoudi et al., 1999), however, R610 in the coding sequence of the peptide KDEL was attached at the C-end of the heavy chain to limit the collected IHH to the Golgi apparatus.

Following the steps of cloning and transfer of plasmids in Agrobacterium Agrobacterium tumefaciens (AGL1) each sheet three tobacco plants Nicotiana benthamiana were infiltrated using a syringe strains of Agrobacterium tumefaciens transformed with plasmids R612 and R610, and incubated in greenhouse conditions for 6 days prior to testing as described in Example 2. After the incubation period, the leaves of each plant (about 20 g biomass) were frozen, chopped, and frozen powder was paremesan to form a homogeneous sample, from which two parts by weight of 1.5 g were taken for extraction (from each plant, Example 3). Content in C5-1 was analyzed for the number of extra the comrade of total protein from each sample using enzyme-linked immunosorbent assay (ELISA) the heavy chain polyclonal immunoglobulin goats against ISM mouse to capture and conjugated with horseradish peroxidase goat immunoglobulin against IHH mouse (H+L) for detection (see Example 3).

As shown in figure 2, agroinfiltration R512 led to the accumulation of 106 mg of antibody per 1 kg wet weight, while the ER-retained form antibodies (R610) to 211 mg/kg wet weight in the same conditions.

As posttranscriptional gene silencing (PDG) has been shown to limit the expression of the transgene in agroinfiltration tobacco plants Nicotiana benthamiana, as well as to demonstrate the anti-co-expression of suppressor HcPro silencing of potato virus Y opposed the specific destruction of the transgene mRNA (Brigneti et al., 1998), contentrate design HcPro (Hamilton et al., 2002) was tested for efficacy in increased expression of C5-1. Coexpressed R612 and R610 with HcPro increased accumulation rates of 5.3 and 3.6 times, respectively, compared with that observed in the absence of HcPro. In the presence of HcPro, controlled by Plastinina expression of C5-1 reached average values of 558 mg/kg wet weight with R612 and 757 mg/kg wet weight with R610 (figure 2). The maximum levels of expression of C5-1 exceeded 1.5 g/kg wet weight (25% of total soluble protein) in some forms of leaves infiltrated as R612 and R610.

To evaluate the scalability of the system expression after agroinfiltration accumulation of C5-1 was subjected to quantitative analysis after conducting the Oia procedure infiltration under vacuum, borrowed from Kapila et al. (1997). In this series of experiments the aboveground part of the plants were infiltrated under vacuum using R612+HcPro or R610+HcPro, after the plants were returned to the greenhouse for 6 days before harvest. With the aim of providing data to provide insight into large-scale production system, the party leaves/stems weighing approximately 250 g from several plants were frozen, chopped in homogeneous samples, and three parts of 7.5 g of material for each batch were selected for analysis. As shown by the results of enzyme-linked immunosorbent assay (ELISA), average levels of accumulation of C5-1 was reached 238 and 328 mg/kg wet weight for infiltrations using R612 and R610, respectively (figure 2).

Example 7. Characteristics of the obtained antibodies

Blot analyses of protein (see Example 3) were used to identify the level of Assembly and fragmentation of antibodies C5-1 IHH in plants producing secreterial (R612) and ER-retention (R610) form of the protein in the experiments with the use of vacuum infiltration and the introduction of the sample syringe. Western blotting with intubation using H+L conjugated to peroxidase immunoglobulin goat against mouse IHH was primarily used to detect the presence of the maximum number of fragments of antibodies, irrespective of their origin is I, on the C5 molecule-1. As shown in figa, all protein extracts that contain fragments of the same molecular size and have the same relative prevalence regardless of the strategy intracellular specify the purpose or method used infiltration. In each case, a large group (>85%), corresponding to the full antibody at about 150 kDa, was displayed with two small groups with a molecular weight of approximately 135 kDa and about 100 kDa, indicating that the antibody accumulated in its fully assembled form (H2L2). Interestingly, fragments of identical electrophoretic mobility was also present in the control IgG, refined out of the line of tumor cells mouse (MORS-21; Sigma #M9269), assuming that the fragments produced in the plant, and the line of mammalian cells were identical and may have arisen as a result of General proteolytic activity. Similar results were obtained using anti-mouse antibodies specific for the heavy chain, for detection.

To verify the identity of the fragments of the antibodies present in the extracts was applied blot analysis of their activity, which is obtained by soaking replica proteins zenderoudi using conjugated to peroxidase IgG human antigen antibodies C. Identity is fully assembled antibodies weight of approximately 150 kDa can be seen on figv. In addition, the fragmentation pattern observed in the Western blot analyses, with the exception of the range with a mass of 100 kDa (see figa), seen in the blot-analysis activity (pigv). Without being bound by theory, this result suggests that the fragment mass 100 kDa does not contain Fab regions of antibodies C5-1, and it can be composed, at least partially, from the heavy chain dimers, the intermediate product of whole antibodies.

Example 8. Purification of the antibody and characterization of the pure product

The antibody was purified from the biomass by only the operation of the chromatographic analysis of the affinity G-protein, and the resulting product was analysed using SDS-page (see Example 4). Stained class "Kumasi" gel on figa shows a large group with a molecular mass of 150 kDa in extracted from the G-protein fractions. This group represents more than 85% of the pure product as in secreted and ER-retained forms, and the content of impurities in both forms of the same (figa, lanes 4 and 5). Western blot analysis by probing polyclonal anti-IHH mice showed that the source of most of the impurities in refined fractions C5-1 is a mouse IHH (data not shown). In reducing conditions were found two the main product with a molecular mass of approximately 25 kDa and 55 kDa, respectively, which corresponds to the molecular weight of the light and heavy chains, respectively (FIGU, track 2). Heavy chain ER-retained antibodies showed a higher mobility than a heavy chain apoplastically antibodies (FIGU, lane 3), which is interpreted as the combined result of adding the amino acid sequence KDEL present at the ends and differences in N-glycosylation due to retention in the ER. On figs shown that purified antibodies (150 kDa) are associated with human IHH, like they do contaminating fragments weighing 75, 90, 100 and 120 kDa, indicating the presence of at least one Fab segment in these fragments. The presence of the Fab segment in the fragment mass 100 kDa contrasted with the result obtained in the analysis of the total extract, in which the group with a mass of 100 kDa was not associated with IHH person. It is assumed that either the number of containing Fab fragments migrating in the total extract with a weight of 100 kDa, was too small to detect during a specified blot analysis activity, or that the fragment migrating at a mass of 100 kDa, consisted of two different molecules, one of them, which is a dimer of a heavy chain (without Fab-segment)and the other containing the antigen.

Reproduction of this system by producing antibodies was evaluated by C the direct comparison of refined products from two different infiltrated parties and three groups of special cleaning of each party. Analysis of SDS-PAGE with staining class "Kumasi" treated groups showed the presence of identical strips in all groups and a significant similarity in the relative distribution (fig.4D).

Example 9. Modification of N-glycosylation of antibodies by coexpressing galactosyltransferase person

To research the possibility of using transient co-expression to control the glycosylation of proteins produced during transient expression, were prepared polygenic expressing clusters for plastocyanin containing the native β1,4 galactosyltransferase (GalT). R622 contained GalT (pigv), and R622 contained the catalytic domain of GalT associated with CTS domain of N-acetylglucosaminyl transferase (GNTI; GNTIGalT (figa). CTS domain of N-acetylglucosaminyl transferase (GNTI) was selected as the "anchor" of the membrane to the catalytic domain of GalT person, while GNTI acts at an early stage of the synthesis of complex N-glican in the ER and the staff of the CIS-Golgi (Saint-Jore-Dupas et al., 2006). In order to avoid limitation by theory, the sequestration activity of GalT at an early stage of maturation of the protein can be expressed in accession β1,4-galactose on ripening glikana and effective inhibition of fokusirovanie and xylotriose kernel. These designs were contentready in plants using a C5-1.

The tobacco plant Nicotiana benthamiana were subjected to the s infiltration (see Example 2) using R612 (secreted form C5-1), R612+R621 (GNT1GalT) or R612+R622 (GalT) in the presence of HcPro. Figure 6 shows the immunological analysis of C5-1, purified from these samples biomass.

Galactosylceramide antibodies was determined by detecting the affinity with agglutinin Erithrina cristagali (ECA)that specifically bind to the β1,4-galactose. As expected, galactose was not detected when C5-1 expressed alone (R612, 6). Galactosylceramide was observed in C5-1, purified from contentrow with R512+R622 (GalT), but not from contentrow with R612+R621 (GNTIGalT, 6). Western blotting performed using antibodies anti-α1,3 fucose, revealed fokusirovanie N-glican on the control C5-1, expressed without galactosyltransferase. Fokusirovanie N-glican was not found on antibody coexpression with GNTIGalT, without regard to the method of agroinfiltration, while coexpressed with native GalT has not led to detectivemisa reduce fokusirovanie antibodies (6). Similar results were obtained with antibodies specific for anti-β1,2-xylose, which showed a complete lack specific to the xylose of immunomagnetic C5-1, coexpression with GNTIGalT, and their presence, when C5-1 was coexpression with GalT (6).

Stained class "Kumasi" gel for direct visual detection completely is designed IHH, and Western blot analysis and blot analysis activity were performed on the same extracts. Based on the data, the expression system antibodies, as described, the amounts of 1.5 g/kg wet weight with more than 85% of the product, consisting of a full length tetramer IHH, a mass of approximately 150 kDa in the total extracts.

Join KDEL peptide to the C-end of the heavy chain was previously used for magnification (2-10X) accumulation of antibodies in helping to remove antibodies from the Golgi apparatus to return to the ER (Schillberg et al., 2003). Using considered in the present description the expression system, the accession KDEL peptide to the heavy chain has doubled the amount of C5-1 output when not used suppressor HcPro silencing. The difference in the amount of C5-1 in the presence or absence of KDEL peptide was significantly reduced when HcPro was used to reduce silencing. ER-retention does not affect the quality, because the fragments, which were observed in the total extracts from plants that produce ER-retained and secreted forms of the antibodies was identical in size and relative distribution.

Antibody C5-1 was isolated on preparative HPLC, and the profile of the N-glican tripticase of glycopeptide EEQNFSTER (SEQ ID NO: 13) antibody C5-1 was analyzed using MALDI-TOF mass spectrometry. As shown in Fi is A, during the expression of C5-1 alone, its population of N-glycans was presented in such complex forms, including ions, consisting of fokusirovannyi and xulosalarining oligosaccharides, as it happened during the observation of the stability of the expression (Bardor et al., 2003). The presence of immature ER-specific glycans, for example, Man-8 and Man-9 may be associated with the protein fraction "en route", as previously reported in the report in respect of the antibodies obtained from plants by transient expression (Sriraman, et al., 2004).

Coexpressed C5-1 with native GalT has led to significant modification of the structure of N-glican, despite the fact that the ions corresponding vysokoopasnym N-picenum remained in excess. The main part of a comprehensive fokusirovannyi and xulosalarining N-glycans (J) disappeared and were found new, partially galactoglucomannan oligosaccharides, some of them are located in specific for plant sozrevaniya and elongation galactose, for example, GalGlcNAcMan3(Xy)GlcNAc2(pigv). Thus, it was demonstrated that coexpression C5-1 with human P1,4 galactosyltransferase caused effective Glyco-engineering of obtaining antibodies from plants.

Coexpressed C5-1 with GNTIGalT have produced a purified preparation of C5-1, where N-glycosylation differed significantly from what was prodem sterowane GalT/C5-1. As shown in figs, galactosylceramide and neglikozilirovannye hybrids (GalGlcNAcMan5GlcNAc2(K) and GalGlcNAcMan5GlcNAc2(G) was present together with the N-glycanase immature oligomannose. Without the need of binding theory, as it is assumed Bakker, et al. (2006), GalGlcNAcMan5GlcNAc2(G) and Man5GlcNAc2(C) may be fragments resulting from the destruction of hybrid GalGlcNAcMan5GlcNAc2endogenous glycosidase, not the intermediate species Mature formation of N-glican. The effect of membrane anchor GNTI was stunning, the antibody C5-1, refined from plants, in which the synthetic enzyme GNTIGalT was transient coexpression with C5-1, contained no traces (≤99%) glycans located in specific plants α1,3 fucose and β1,2-xylose, demonstrating that the full modification fokusirovanie and xylotriose was reached during the transient co-expression.

All opposed to in the description of the materials included in it by reference.

The description of the present invention has been given with the involvement of one or more variants of its implementation. However, for the specialist in this field of knowledge will be obvious that it can be made various changes and modifications of the invention without going beyond the scope of the invention, characterized by the claims.

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1. The method of synthesis of the target protein with reduced kilogramovaya, low fokusirovanie, or a combination comprising introducing into the plant, plant part or plant cell a nucleotide sequence at 80-100 % identical to the nucleotide sequence defined in SEQ ID NO: 17, and encodes a protein compound containing cytoplasmic tail segment, a transmembrane domain, a stem region (CTS domain of N-acetylglucosaminyltransferase (GNT1), fused with the catalytic domain of the beta-1,4-galactosyltransferase (GalT), and the specified first nucleotide sequence is functionally linked to a first regulatory region that is active in the plant; and the second nucleotide sequence for encoding the target protein, PR is than the specified second nucleotide sequence is functionally linked to a second regulatory region, which is active in the plant, as well as the transient coexpression the first and second nucleotide sequences with the synthesis of the target protein containing glikana, reduced kilogramovaya, low fokusirovanie or their combination when compared with the same target protein, derived from wild plants.

2. The method according to claim 1, in which the regulatory region is the first promoter of plastocyanin or the first 35S promoter, and a second regulatory region is the second promoter of plastocyanin or second promoter 35S.

3. The method according to claim 1, which further third nucleotide sequence is introduced into the plant, and the third nucleotide sequence encodes a suppressor of silencing and functionally connected with the third regulatory region that is active in the plant.

4. The method according to claim 3, in which a third regulatory region is a promoter of plastocyanin or 35S promoter.

5. The method according to claim 2, in which the addition of the third nucleotide sequence is introduced into the plant, and the third nucleotide sequence encodes a suppressor of silencing and functionally connected with the third regulatory region that is active in the plant.

6. The method according to claim 5, in which the third promoter regulatory region is a promoter of plastocyanin is whether the 35S promoter.

7. The method according to claim 1, in which the target protein is an antibody or antigen.

8. The method according to claim 7, in which the second nucleotide sequence encoding a target protein, contains a nucleotide sequence encoding a light chain, functionally associated with a regulatory region that is active within plants, and the nucleotide sequence encoding a heavy chain, functionally associated with a regulatory region that is active in the plant, and the product of the nucleotide sequence that encodes a light chain and a nucleotide sequence that encodes a heavy chain combined with the formation of antibodies.

9. The method according to claim 8, in which the regulatory region is a promoter of plastocyanin.

10. Nucleic acid, essentially consisting of a nucleotide sequence of from 1 to 1062 presented in SEQ ID NO: 17 (GNT1-GalT), or essentially consisting of a nucleotide sequence that exhibits from 80 to 100% identity with nucleotides 1 to 1062 sequence SEQ ID NO: 17), where the nucleotide sequence encodes a protein that modifies glycosylation of a target protein.

11. Composite protein GNT1-GalT, designed to modify the glycosylation of a target protein containing a CTS domain of N-acetylglucosaminyltransferase, merged with the catalytic domain is m beta-1,4-galactosyltransferase, moreover, an integral protein contains amino acids 1 to 354 of the sequence SEQ ID NO: 18.

12. Nucleic acid encoding a protein compound according to item 11.

13. The plant is designed for expression of a protein that modifies glycosylation of a target protein containing nucleic acid for PP, 12 or a protein according to item 11.

14. Plant cell used for expression of a protein that modifies glycosylation of a target protein containing nucleic acid for PP, 12 or a protein according to item 11.

15. The seed used for expression of a protein that modifies glycosylation of a target protein containing nucleic acid for PP, 12 or a protein according to item 11.

16. The method according to claim 3, in which the target protein is an antibody and synthesized in the amount of up to 1.5 g / kg raw leaves.

17. The method according to claim 1, in which the synthesized target protein is not fokusirovannym and/or xylopyranosyl.

18. The method according to claim 1, in which the synthesized target protein is 5% less fokusirovannym and xylopyranosyl compared with the same protein obtained in the wild type plant.

19. The method according to claim 1, in which the synthesized target protein is 2% less fokusirovannym and xylopyranosyl compared with the same protein obtained in the wild type plant.

20. The method according to claim 1, in which ne is the first nucleotide sequence comprises nucleotides 1-1062 sequence SEQ ID NO: 17 (GNT1-GalT).



 

Same patents:

FIELD: biotechnologies.

SUBSTANCE: invention proposes variable domains of heavy (VH) and light (VL) chains of murine antibody against tumour necrosis factor alpha (TNF-α) of a human being, as well as antigen-binding fragment Fab, which are selectively bound to TNF-α of the human being and neutralise it.

EFFECT: invention can be further used in development of medicines for therapy of TNF-α-mediated diseases and for diagnostics of such diseases.

3 cl, 5 tbl, 7 ex

FIELD: biotechnologies.

SUBSTANCE: invention relates to a molecule of nucleic acid, which is a cyclic or a linear vector fit for expression, of at least one target polypeptide in cells of mammals, including (a) at least one expressing cassette (POI) for expression of the target polypeptide; (b) an expressing cassette (MSM), including a gene of a selective marker of mammals; (c) an expressing cassette (MASM), including an amplificated gene of a selective marker of mammals; besides, the expressing cassette (POI) is flanked in direction 5' by the expression cassette (MASM), the expression cassette (MSM) is localised in direction 3' from the expression cassette (POI) and in which the expression cassettes (MASM), (POI) and (MSM) are arranged in the same orientation from 5' to 3'. Also the method is disclosed to produce the specified molecule of nucleic acid of the vector, as well as a cell of a host mammal, containing the specified molecule of nucleic acid of the vector, the method to produce a host cell containing the specified molecule of nucleic acid of the vector, and also the method to produce the target polypeptide, using the specified host cell.

EFFECT: invention makes it possible to efficiently produce a target polypeptide in mammal cells.

24 cl, 2 dwg, 4 tbl, 13 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. There are presented: a method for tumour cell growth inhibition in an individual and a method for immune response enhancement in an individual involving the introduction of a PD-1 monoclonal antibody and a CTLA-4 10D1 monoclonal antibody to the individual. The PD-1 monoclonal antibody has the following properties: it binds to human PD-1 having the value KD equal to 1×10-8 M or less; however it binds neither to human CD28, nor to CTLA-4, nor to ICOS; it is able to enhance the T-cell proliferation in the mixed lymphocyte reaction (MLR) analysis; it is able to enhance the gamma interferon production in the MLR analysis; it is able to enhance the interleukine-2 (IL-2) secretion in the MLR analysis.

EFFECT: invention provides a synergic effect when using the above antibodies in a combination.

4 cl, 54 dwg, 7 tbl, 25 ex

Novel antibodies // 2490277

FIELD: chemistry.

SUBSTANCE: present invention relates to immunology. Disclosed is an anti-α5β1 antibody, which is described through amino acid sequences of six hypervariable regions and an antigen-binding moiety thereof. Described are conjugates of the disclosed antibodies with a medicinal agent or a label, a pharmaceutical composition, use of the disclosed antibodies to prepare a medicinal agent, methods and an industrial product for inhibiting angiogenesis and/or vascular permeability in a subject, and for treating cancer, an ophthalmic disease and an autoimmune disease in a subject. The invention describes an isolated nucleic acid, an expression vector, a cell and a method of producing an antibody or an antigen-binding moiety thereof, as well as a method of detecting α5β1 protein in a sample.

EFFECT: present invention can find further use in therapy and diagnosis of α5β1-mediated diseases.

52 cl, 11 dwg, 6 ex

FIELD: chemistry.

SUBSTANCE: disclosed are versions of human IL13 specific antibodies and a producing hybridoma cell line deposited in ATCC under number PTA-5657. Described are: versions of encoding polynucleotides; an antibody expression vector; a host cell for antibody expression, as well as versions of a method of producing an antibody using a vector, polynucleotide, hybridoma or host cell. The invention discloses a pharmaceutical composition for treating IL13-mediated diseases and methods of treating allergic, inflammatory and other diseases, which employ an anti-IL13 antibody.

EFFECT: providing antibodies which do not bind with target IL13 and neutralise activity of human IL13.

39 cl, 29 dwg, 11 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, more specifically to expression constructs, and may be used for immunoglobulin expression. An expression vector contains one open reading frame (sORF) insert which contains a first sequence of nucleic acid coding a first polypeptide; a first intermediate sequence of nucleic acid coding a first protein cleavage site containing an autoprocessing element with an intein segment providing proteolytic sORF polypeptide cleavage between the first polypeptide and the intein segment and the second polypeptide, but not ligation of said first polypeptide with said second polypeptide; and a second sequence of nucleic acid coding the second polypeptide. The expression vector is able to express a mammalian polypeptide coding sORF and cleaved in said first protein cleavage site in a host cell; consisting of the first polypeptide - an immunoglobulin heavy chain, and the second polypeptide - an immunoglobulin light chain able to be assembled into a multimer.

EFFECT: invention provides functional antibody production with 'correct' setup and assembly.

40 cl, 9 dwg, 57 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and can be used to obtain monoclonal antibodies against the Yersinia pestis V antigen. The strain of hybrid animal cells Mus musculus 2B8 is obtained by immunising BALB/c mice. The mice are immunised by four-time administration of a recombinant V antigen in a dose of 100 mcg/mouse. On the third day after the last immunication, splenocytes of immune mice (1×108 cells) are hybridised with mouse myeloma cells RZ-X63 Ag/8-653 (1×107 cells). The fusion agent used is polyethylene glycol (Sigma, USA). Hybridisation is followed by selection, screening, cloning and cryopreservation of the hybridoma. The strain is deposited in the state collection of pathogenic microorganisms and cell cultures (GKPM-Obolensk) under number N-20.

EFFECT: strain of hybrid cultured cells, which produces monoclonal antibodies which are specific to the Y pestis V antigen, is suitable for constructing test systems for detecting plague pathogens.

8 dwg, 3 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and can be used to obtain monoclonal antibodies against the Yersinia pestis V antigen. The strain of hybrid animal cells Mus musculus 5G6 is obtained by immunising BALB/c mice. The mice are immunised by four-time administration of a recombinant V antigen in a dose of 100 mcg/mouse. On the third day after the last immunication, splenocytes of immune mice (1×108 cells) are hybridised with mouse myeloma cells RZ-X63 Ag/8-653 (1×107 cells). The fusion agent used is polyethylene glycol (Sigma, USA). Hybridisation is followed by selection, screening, cloning and cryopreservation of the hybridoma. The strain is deposited in the state collection of pathogenic microorganisms and cell cultures (GKPM-Obolensk) under number N-19.

EFFECT: strain of hybrid cultured cells, which produces monoclonal antibodies which are specific to the Y pestis V antigen, is suitable for constructing test systems for detecting plague pathogens.

8 dwg, 2 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: invention presents a method for characterising a light chain type in a composition of recombinant polyclonal antibodies which may be used for assessment of various antibodies produced by a polyclonal cell line in the process of production, as well as batch consistency of the antibodies found in the polyclonal products. A method for structural characterisation is based on heavy chain removal and residual light chain separation by chromatographic separation and mass spectrometry of intact light chain types. What is also disclosed is a method for detecting the presence of versions in intact light chain populations in two or more compositions of the recombinant polyclonal antibodies prepared of one polyclonal cell culture in different moments of time in the process of cultivation, or of various polyclonal cell cultures in a specific moment of time.

EFFECT: use of the invention enables making the specific drug batch complying with the pre-set production specification.

19 cl, 8 dwg, 2 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to antibody crystallisation. What is presented is a method for series crystallisation of hIL-12 specific antibody ABT-874. It enables producing an aqueous crystallisation mixture of the antibody of the concentration of approximately 0.5 to approximately 280 mcg/ml and polyalkylene glycol of average molecular weight approximately 400-10000 and of the concentration of 5% to 30% (wt/vol.). The prepared mixture is incubated at pH approximately 4 to approximately 6.5 and at temperature approximately 4 °C to 37 °C for form the crystals 2-500 mcm long. There are described versions of the hIL-12 crystals prepared by said method. There are disclosed versions of the pharmaceutical composition, an injected liquid composition, a crystalline suspension composition, versions of a method of treating the hIL-12 mediated disorders, based on using the antibody crystals. What is presented is using the crystals for preparing the pharmaceutical composition for treating IL-12 associated disorder.

EFFECT: using the invention enables producing the antibody crystals on a commercial scale that can find application for industrial production of the antibody crystals for treating the hIL-12 mediated disorders.

40 cl, 13 dwg, 3 tbl, 47 ex

Vns-met-histones // 2498997

FIELD: biotechnologies.

SUBSTANCE: nucleic acid molecule codes a polypeptide consisting of two residues of methionine as the first and the second N-end amino-acid residues connected through a peptide link to a mature eucariotic histone. Polypeptide is obtained by cultivation of a host cell transformed by an expression vector including the above molecule of nucleic acid. Polypeptide is used as part of pharmaceutical composition for therapy of cancer, bacterial, virus or fusarium infections. Besides, polypeptide is used as part of composition for diagnostics of a patient in relation to response to pharmaceutical composition containing the above polypeptide, or in relation to curability using it.

EFFECT: invention allows improving efficiency of recombinant expression and simplifying determination of the above polypeptide in presence of endogenic histones at preservation of biologic activity of mature eucariotic histone.

17 cl, 3 dwg, 6 tbl, 7 ex

FIELD: biotechnologies.

SUBSTANCE: invention proposes compounds of labyrinthpeptins A1, A2, or A3 of formula (I) , where {A}, {B}, {C}, R1-R6, m and n have the values specified in the formula of the invention. Compounds are obtained at fermentation of Actinomadura namibiensis DSM 6313 strain under acceptable conditions in cultural environment till one or more compounds of formula (I) are formed. The invention proposes the deoxyribonucleic acid (DNA) coding preprolabyrinthpeptin A2, and the deoxyribonucleic acid (DNA) coding preprolabyrinthpeptin A1, as well as preprolabyrinthpeptins A1 and A2, and prolabyrinthpeptins A1 and A2. Labyrinthpeptins of formula (I) are used for therapy of infections caused by gram-positive bacteria, virus infections and/or neuropathic pain caused by inflammation.

EFFECT: improving therapy efficiency.

24 cl, 4 tbl, 20 ex

FIELD: biotechnology.

SUBSTANCE: protease is presented which has enhanced milk clotting activity, containing amino acid sequence at least 80 % identical to SEQ ID NO: 3, where the said protease has at least one mutation selected from the group consisting of: (a) substitution of glutamine, corresponding to glutamine at a position of 265 in SEQ ID NO: 3, with amino acid; and (b) replacement of glutamine, corresponding to glutamine at a position of 266 in SEQ ID NO: 3, with amino acid. DNA is described which encodes the said protease, the expression vector containing the said DNA, and the cell transformed with the said vector, designed for expression of the said protease. The method of production of protease having enhanced milk clotting activity is proposed, comprising culturing the said transformed cell in the cultural medium and isolation of protease from the cultural medium.

EFFECT: invention enables to obtain the protease with enhanced milk clotting activity.

16 cl, 2 dwg, 4 tbl

FIELD: biotechnologies.

SUBSTANCE: method includes a stage of yeast cultivation, transformed by a vector containing a DNA sequence, determined by the formula X-B-Y-A, coding the precursor of insulin glargine, where X is a sequence of leader peptide, containing at least one amino acid. B is a B1-B30 sequence of amino acids of B-chain of the insulin glargine molecule. Y is a linker peptide containing at least two amino acids. A is an A1-A21 sequence of amino acids of an A-chain of a molecule insulin glargine, a stage of extraction of an expressing precursor of insulin glargine, a stage of crystallisation of the extracted precursor of insulin glargine, a stage of completion of fermentative conversion of insulin glargine precursor crystals at pH from 8 to 10 in presence of tripsin or tripsin-like ferment and water soluble organic dissolvents at the ratio from 40% to 60% of the reaction mix with formation of insulin glargine, containing at least one related admixture. Then the stage of insulin glargine treatment by reverse phase highly efficient liquid chromatography is carried out on a chromatographic matrix, using a polar organic buffer dissolvent in a water phase, containing a buffer based on organic acid, in which the matrix is first balanced with 10% acetonitrile in 250 mM of acetic acid with further elution of insulin glargine in the specified acetonitrile. Then the matrix is again balanced with 10% acetonitrile in the buffer on the basis of organic acid in concentration from 20 mM to 200 mM at pH from 3 to 8.5 with subsequent elution of insulin glargine in the specified acetonitrile, and further repeatedly the matrix is balanced with 6% ethanol in the buffer on the basis of organic acid in concentration from 10 mM to 50 mM with subsequent elution of the specified insulin glargine in the specified ethanol. Further the treated insulin glargine is deposited by means of addition of the buffer on the basis of citric acid and zinc chloride at pH from 6 to 8.

EFFECT: invention makes it possible to produce insulin glargine with high purity and low content of glycolised admixtures.

12 cl, 9 dwg, 8 tbl, 9 ex

FIELD: biotechnologies.

SUBSTANCE: plasmid genetic structure pOL-DsRed2 is produced, being built on the basis of a plasmid vector pIRES (Clontech), where fragments of cDNA of human genes OCT4 and LIN28 are placed, being connected with a nucleotide sequence coding P2A-peptide and gene cDNA, coding fluorescent protein DsRed2.

EFFECT: invention provides for simultaneous translation of human proteins OCT4 and LIN28 and fluorescent protein DsRed2 in production of induced pluripotent stem cells of a human being and animals in medicine and veterinary science.

3 cl, 1 dwg, 1 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry. Disclosed is a method of isolating and purifying recombinant human growth hormone which is secreted by Saccharomyces cerevisiae yeast during fermentation thereof in suitable conditions. The target protein is precipitated in biomass-free culture fluid by either acidification to pH 2.9-4.0 or adding polyethylene glycol with molecular weight of 3000-6000 Da. The obtained precipitate is then dissolved in a suitable solvent. Preliminary purification of the target protein is carried out either by anion-exchange chromatography at pH 5.6 or by diafiltration in the presence of 0.1-0.5 M sodium chloride. Main purification of the target protein is then carried out by anion-exchange chromatography at pH not below 7.3 and gel filtration.

EFFECT: invention enables to obtain a growth hormone which is free from parent proteins, host-producer protein and other impurities such as pigments, with output of up to 60%.

8 ex

FIELD: biotechnology.

SUBSTANCE: method provides for culturing mammalian cells expressing the recombinant protein of interest in a culture medium containing anti-aging compound selected from carnosine, acetyl-carnosine, homo-carnosine, anserine, and K-alanine and their combinations, under conditions and for a time sufficient for expression of the protein.

EFFECT: invention enables to improve the performance of the cell culture selected from higher titer, of increased specific cell productivity, increased cell viability, increased integrated viable cell density, decreased accumulation of high molecular aggregates, decreased accumulation of acidic molecules, and their combinations.

44 cl, 14 dwg, 3 ex

FIELD: biotechnologies.

SUBSTANCE: method consists in expression of a gene of a human plasminogen fragment from 453 to 543 amino acid within E.coli cells with subsequent extraction and treatment of a finished product from cell periplasm. Expression is carried out in cells E.coli BL21 (DE3), transformed by plasmid DNA pEK5 or pEK5H with physial maps represented in figure 2, containing a gene of target polypeptide fused with a gene of signal peptide OmpA, origin of replication pUC ori, a gene of resistance to kanamycin and a gene coding lac-repressor under control of T7-promotor. At the same time the recombinant plasmid DNA pEK5H additionally contains between genes OmpA and target polypeptide the sections coding amino acid sequences HHHHHH and DDDDK.

EFFECT: invention provides for secretion of target polypeptide into cell periplasm with high yield.

4 cl, 5 dwg, 6 ex

FIELD: biotechnologies.

SUBSTANCE: fusion peptide is presented for neutralisation and destruction of organophosphorous compounds, comprising a signal peptide TAT3 with amino acid sequence SEQ ID NO: 5, presented in the description, functionally connected to the sequence of organophosphate hydrolase SEQ ID NO: 18, classified as protein EC 3.1.8. The following components are described: extracted polynucleotide, which codes the specified fusion protein; a vector containing the specified polynucleotide; and a procaryotic host cell containing the specified vector and expressing the specified fusion protein. The method is proposed to produce a ferment, which destroys organophosphorous compounds, including expression of the specified polynucleotide in the procaryotic host cell and production of the specified ferment.

EFFECT: invention makes it possible to increase expression of organophosphate hydrolase in a host cell.

13 cl, 1 tbl, 9 dwg, 2 ex

FIELD: biotechnology.

SUBSTANCE: method of highly effective production of methionine-containing form of staphylokinase (SAK-1) and some of its biologically active analogs in a host cell, providing culturing a bacterial cell transformed by the expression vector which comprises the DNA sequence encoding SAK-1 at a high degree of aeration and reducing the level of oxygen by 5% of atmospheric level with achievement the exponential growth phase.

EFFECT: use of the invention provides a significant increase in the yield of the target product.

11 dwg, 4 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to a method of preparing a pharmaceutical composition, CHO cell to obtain the desired protein, the CHO cell - DNA recipient encoding the desired polypeptide, the method of production of the desired polypeptide. The method of production of the desired polypeptide comprises cultivation of CHO cell which is transformed DNA encoding alanine aminotransferase and DNA encoding the desired polypeptide. In the particular case the CHO cell is cultured in α-ketoglutarat-containing medium. The method of preparing of the pharmaceutical composition comprises preparing of the desired polypeptide with the method described above. The polypeptide is mixed with pharmaceutically acceptable carriers or additives. The preparation is prepared. The CHO cell for preparing of the desired protein has DNA transferred into it, encoding alanine aminotransferase, and DNA transferred into it, encoding the desired polypeptide.

EFFECT: invention enables to prepare a desired polypeptide with a high yield.

10 cl, 22 dwg, 3 ex

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