Recombinant dna, which codes functionally active hybrid protein gl7aca-acylase with chitin-binding domain (brdgl7aca-cbd) and recombinant plasmid psvh0108, providing for its synthesis in cells escherichia coli, recombinant strain escherichia coli bl21(de3)/psvh0108-producer brdgl7aca-cbd

FIELD: biotechnologies.

SUBSTANCE: recombinant DNA is produced, which codes functionally active hybrid protein (BrdGl7ACA-cbd), consisting of amino-acid sequence of acylase glutaryl-7- aminocephalosporanic acid of strain Brevundimonas diminuta All-Russian collection of industrial microorganisms B-1297 and chitin-binding domain of chitinase Al Bacillus circulans. Recombinant plasmid pSVH0108 is constructed for expression of BrdGl7ACA-cbd in cells E.coli, containing sequence of recombinant DNA that codes hybrid protein under control of promotor and terminator of RNA-polymerase of phage T7. As a result of E.coli strain tranformation with this recombinant plasmid and selection of transformed clones, a new strain E.coli BL21(DE3)/pSVH0108 cbd is produced - producer of hybrid protein BrdGl7ACA-cbd.

EFFECT: high yield of recombinant ferment.

4 dwg, 2 tbl, 7 ex


The present invention relates to the field of biotechnology, in particular genetic engineering, and can be used in microbiological industry for preparation of semisynthetic cephalosporin antibiotics of the new generation. Available:

recombinant DNA encoding a functionally active hybrid protein BrdGl7ACA-cbd, which consists of the acylase glutaryl-7-aminocephalosporanic acid strain Brevundimonas diminuta VKM B-1297 and chitin-binding domain of chitinase A1 of Bacillus circulans;

recombinant plasmid pSVH0108 containing a DNA fragment which encodes the complete amino acid sequence BrdGl7ACA-cbd, and providing a high level of expression of a named hybrid protein in Escherichia coli cells;

- recombinant strain E. coli BL21(DE3)/pSVH0108 producing a hybrid protein BrdGl7ACA-cbd.

The level of technology

Gl7ACA-acylase [1] belong to an extensive family of penicillin-amidase (EC, capable of converting the penicillins and cephalosporins in valuable intermediate compounds used in the production of semisynthetic beta-lactam antibiotics of the new generation [2, 3].

Due to the need for increased production of this now widely used group of antibiotics [4-6] improvement of the known methods of obtaining the necessary for their synthesis enzymes, including by those who of ology recombinant DNA, is of great practical interest.

To date, the known primary structure of bacterial genes Gl7ACA-acilis from various strains of Pseudomonas; described recombinant plasmid DNA for synthesis of these enzymes in the cells of E. coli and Bacillus subtilis, as well as the methods of production of recombinant forms of the enzymes with their use [1, 7-12].

The General disadvantages of the known methods heterologous expression Gl7ACA-Atzilut are the low level of synthesis of the recombinant product [7], the complexity of processes scale fermentation strains [8-10] and release derived drugs Gl7ACA-acilis from impurities, in particular from non-specific beta-lactamase [5].

Eliminate these drawbacks can be achieved on the development of new systems of expression Gl7ACA-acylase, providing higher yield of active enzyme and simplification of the methods of its treatment. As one of the most effective ways to purify the protein is affinity chromatography [13], in recent years, widespread expression systems for production of recombinant protein products in the form of hybrid proteins containing auxiliary polypeptide specifically binding to a compound, which is used as a ligand in the subsequent protein purification by affinity chromatography. Receipt the hybrid proteins although it is quite simple and effective method, is associated with some difficulties, one of which is the possibility of partial or total loss of activity due to modification of the structure of protein molecules after binding it with auxiliary polypeptide. In such situations, it may be provided by subsequent cleavage of the hybrid protein (extra stage) on the components under the action of chemical or enzymatic agents, however, the introduction of the procedure of splitting the expressed hybrid protein requires subsequent additional purification.

Specific features of enzymes in the form of a hybrid protein is the principal point is the search for combinations of target and auxiliary protein, in which the presence of an additional component in the preparation of the enzyme will not adversely affect the properties of the target product, and the Association intended for purification of the polypeptide of interest protein, in turn, will lead to a decrease in its specific binding activity. Design of a hybrid protein with such properties simplifies the purification of the target product and guarantees the full preservation of its activity in the composition of the fused protein. With the right choice of an additional component of recombinant enzymes in g is Britney constructions are fully prepared for use as immobilized drug if it is provided by process.

As for Gl7ACA-acylase, attempts to carry out her expression in the form of a fused protein is essentially limited by derivatization bearing 6 additional N-terminal his-tag residues [14], which are traditionally used for subsequent chromatographic purification of many recombinant proteins. However, sorbents for His-tag forms, as a rule, are quite expensive and not suitable for subsequent direct use of biocatalysts, which significantly limits their practical application.

In the present invention was tasked with the expression of a hybrid protein comprising Gl7ACA-acylase, which would fully preserved functional activity of both its components and its immobilized form could be directly applied in the process of biotransformation, as well as the development of vectors and systems necessary for its receipt.

Disclosure of inventions

The task of obtaining any functionally active hybrid protein comprises the following steps:

a) the design of the structure gene of the hybrid protein, suggesting optimal from the point of view of preserving the functional activity of the combination of the target and auxiliary protein; b) obtaining and combining with p the power of the methods of recombinant DNA nucleotide sequences, encoding components of the hybrid protein; C) selecting host cells and the creation of vector constructs for expression of the hybrid protein in a selected heterologous system; d) optimization of conditions for the production and purification of the hybrid protein.

The goal is to obtain functionally active hybrid protein-based Gl7ACA-acylase, suitable for subsequent use as immobilized drug, due to the fact that

1) the proposed recombinant DNA encoding a hybrid protein BrdGl7ACA-cbd, in which the C-terminal region of the sequence of the alpha-subunit of the acylase glutaryl-7-aminocephalosporanic acid strain Brevundimonas diminuta VKM B-1297 embedded chitin-binding domain of chitinase A1 of Bacillus circulans;

2) constructed recombinant plasmid pSVH0108 containing a recombinant DNA sequence which encodes a hybrid protein BrdGl7ACA-cbd, and provides its synthesis in cells of Escherichia coli with high yield;

3) the transformation expressing the plasmid pSVH0108 cells of the strain E. coli BL21(DE3) were obtained recombinant Escherichia coli strain BL21(DE3)/pSVH0108 characterized by a high level of induced synthesis and stable production of a hybrid protein BrdGl7ACA-cbd, in which the activity retain both its constituent protein domain.

The choice of the chitin-binding domain of chitinase A1 of Bacillus circulans CBD) as an auxiliary polypeptide was due to the fact, that chitin (chitin media)with which it interacts, has a natural origin, is cheaper compared to many other media for affinity chromatography, and the relationship that forms including D hybrid protein with the media, different physical and chemical stability, it can be used multiple times.

In addition, in certain cases the use of chitin-binding domains in combination with the active protein, they usually did not adversely affect the function of the active component.

Gl7ACA-acylase are heterotetramer composed of 2 α and 2 β subunits [15], which are formed from a single polypeptide precursor. Complete coding sequence of a gene BrdGl7ACA-acylase (SEQ ID No. 5) was obtained by polymerase chain reaction (PCR) using as the template the chromosomal DNA isolated from strain Brevundimonas diminuta VKM B-1297, and as primers synthetic oligonucleotides from nucleotide sequences SEQ ID No. 1 (primer 7_ACA_F) and SEQ ID No. 2 (primer 7_ACA_R). This sequence was introduced into the vector Rast (figure 1), derived from vectors 21d and rsus (see example 2), to obtain the recombinant plasmids pSVH1811 (see example 3).

The nucleotide sequence encoding the chitin-svyazyvayus the domain of chitinase A1 of Bacillus circulans (SEQ ID No. 15), received from the commercial plasmid pTYB4 (New England Biolabs.)

The position of the CBD in the structure of the hybrid protein was determined on the basis of data on the spatial structure Gl7ACA-acylase Brevundimoinas diminuta, complexes of the enzyme with the substrate and product of the reaction and the properties dvukhzaryadnykh periplasmatic enzymes that undergo autocatalytic processing[9, 15, 16].

Conducted design modifications of the end sections of polypeptide chain subunits showed that the preferred site for the binding of CBD is the C-terminal region of the alpha - subunit Gl7ACA-acylase, because it is at a remote distance from the active center of the enzyme and does not participate in catalysis. Although there is currently no clear views on the mechanism of removal of spacer elements peptide [15, 17], there is reason to believe that the correct cleavage of the alpha-subunit and peptide spacer elements important to the safety of the C-terminal amino acids of the alpha-subunit, therefore, to reduce the likelihood of detachment of CBD together with the spacer elements of the plot seemed appropriate to include the sequence of the chitin-binding domain so that after him was a few amino acids, the end of the alpha-subunit. Accordingly, the position of the CBD in the structure of the hybrid protein was identified between amino acids R18 and T185, which assumed the embedding sequence that encodes a chitin-binding domain, between nucleotides 551 and 554 gene BrdGl7ACA-acylase (SEQ ID No. 5).

The Union of the sequence, the coding CBD (SEQ ID No. 15), with the sequence of a gene acylase was held in the vector, which was previously included gene BrdGl7ACA-acylase.

To create the expression vector directing in E.coli cells the synthesis of a hybrid protein, based plasmids pSVH1811 was constructed intermediate vector pSVH0107 obtained by introducing into it an additional restriction site Vne1 in position 551-554 SEQ ID No. 5. This site was then used for embedding in a specified position of the gene coding sequence of the chitin-binding domain (CBD). Obtained by expressing this recombinant plasmid for synthesis of a hybrid protein BrdGl7ACA-cbd was identified as pSVH0108 (figure 2).

Comparison of amino acid sequences BrdGl7ACA-cbd, BrdGl7ACA-Out sequence BrdGl7ACA-acylase is shown in figure 3.

By transforming cells of Escherichia coli strain BL21(DE3) [21] constructed a plasmid pSVH0108, selection and cultivation of clones transformed with high-level synthesis of functionally active hybrid protein obtained recombinant Escherichia coli strain BL21(DE3)/ pSVH0108 producing a hybrid protein consisting of BrdGl7ACA-acylase, containing the her C-terminal region sequence of the alpha-subunit of chitin-binding domain of chitinase A1 of Bacillus circulans. Synthesis BrdGl7ACA-cbd in the resulting recombinant strain is under cultivation in conventional selective media with the addition of the inducer isopropyl-D-thiogalactoside (IPTG) or lactose.

Thus, the present invention includes three objects:

The first object is a recombinant DNA that encodes a hybrid protein BrdGl7ACA-cbd, consisting of the amino acid sequence of the acylase glutaryl-7 - aminocephalosporanic acid strain Brevundimonas diminuta VKM B-1297 and chitin-binding domain of chitinase A1 of Bacillus circulans, and characterized by nucleotide sequence SEQ ID No. 16.

A second object of the recombinant plasmid pSVH0108 for synthesis of a hybrid protein BrdGl7ACA-cbd in Escherichia coli cells, which are formed by the vector rst consisting of a fragment of the modified area polylinker plasmids 21d containing the promoter and terminator of RNA polymerase of phage T7, divided by the area of polylinker, and fragment of plasmid rsus containing the replicon RA and the gene for resistance to kanamycin, and the sequence of the recombinant DNA according to claim 1, integrated in polyglycerol the region of the specified vector.

The third object is a recombinant Escherichia coli strain BL21 (DE3)/pSVH0108 producing a hybrid protein BrdGl7ACA-cbd.

Brief description of drawings

Figure 1 - physical and genetic map of the vector pACT7. The indicated position of the indicator with itov restriction, lots of promoter and terminator RNA polymerase of phage T7 (T7 prom and T7 term), the starting area replication (p15Aori), the gene for resistance to kanamycin (denoted by KN(R), filling black).

Figure 2 - physical and genetic map of the plasmid pSVH0108. The position of the gene BrdGl7ACA (filled gray), the position of the chitin binding domain (filling stroke), and the remaining notation is as in figure 1.

Figure 3 - comparison of amino acid sequences BrdGl7ACA-cbd, BrdGl7ACA-Out sequence BrdGl7ACA-acylase. Black selected sequence corresponding to the C-end of the alpha-subunit, underlined sequence peptide spacer elements, italic - CBD-domain frame - rjlbhe.ofz sequence N-Terminus of the beta subunit.

Figure 4 - electrophoregram separation in 12% of the LTO-page of fractions obtained during the purification of the recombinant protein BrdGl7ACA-cbd. Tracks: 1 - marker, mol. weight, 2 - fraction after washing the column with hainesville granules after immobilization of the protein BrdGl7ACA-cbd, line 3 - purified product BrdGl7ACA-cbd.

The implementation of the invention

When carrying out the invention in addition to the methods disclosed in detail in the following examples used are well known in the art the techniques described in the manuals of molecular biology and genetic engineering [19, 20].

Example 1. The selection gene Gl7ACA-acylase

The primary structure of the genes G7ACA-Atzilut, isolated from various strains of Pseudomonas and Brevundimonas known (see access numbers in GenBank AAC34685, AAN39264, AAP68796). These sequences have a high degree of conservatism, which greatly simplifies the task of selection gene Gl7ACA-acylase by PCR amplification.

As a matrix to obtain gene Gl7ACA-acylase using chromosomal DNA of strain Brevundiminas diminuta VKM B-1297, and as primers the oligonucleotides 7ACA_F (SEQ ID No. 1) and 7ACA_R (SEQ ID No. 2), specific to the conservative sites flanking the full coding sequences of known genes Gl7ACA-Atzilut.

1 µg of genomic DNA Brevundiminas diminuta VKM B-1297, highlighted by traditional methods, are denatured by heating at 100° for 5 minutes, placed on ice and subjected to 30 cycles of PCR using a set of Expand Long Template PCR system (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions.

Mixture for PCR (50 ál):

5 ál of 10x PCR buffer (Roche Diagnostics GmbH");

5 ál of genomic DNA (200 ng/ál);

5 μl of 3 μm primer 7ACA_F;

5 μl of 3 μm primer 7ACA_R;

5 μl of 2.5 mm dNTP each type;

25 µl of deionized water;

1 μl of DNA polymerase (Roche Diagnostics GmbH).

Conditions for PCR: 94°, 5' (denaturation), 94°, 30"; 50°, 30"; 72°,2' (amplification).

After amplification, 5 µl of the PCR mixture is analyzed by electrophoresis in 1% agarose gel and identify homogeneous fragment p is Merom about 2.2 TPN. Fragment isolated from the gel using the set Wizard PCR Preps Kit (Promega, USA) according to the manufacturer's instructions and subjected to automated sequencing on the device ABIPrizm 3100 DNA Sequencer using primers BRD1 (SEQ ID No. 3), BRD2 (SEQ ID No. 4), 7ACA_F, 7ACA_R.

The comparison of the obtained nucleotide sequences from the GenBank database using the BLAST program showed that it possesses a high degree of homology with the corresponding coding sequences of other bacterial Gl7AC-Atzilut. On this basis it was concluded that this fragment corresponds to the gene Gl7ACA-acylase strain Brevundimonas diminuta VKM B-1297. Installed the nucleotide sequence of the gene BrdGl7ACA-acylase given in the list under the number SEQ ID No. 5.

Example 2. Design vector-media pACT7

Vectors based on the promoter of T7 phage provide high expression of heterologous genes in E. coli strains synthesizing T7 polymerase, and commercially available [21]. At the same time, preparations Gl7ACA-acilis used for biotransformation of cephalosporin antibiotics, should not contain impurities of beta-lactamases, genes which are present in most commercial vector designs of this type. Therefore, for the expression of BrdGl7ACA-acylase and other biotransformation enzymes antibiotics, free from impurities of beta-lactamases, it is advisable IP is to alsowhat vectors, bearing markers plasmid stability, non-AmpR. Such vectors also have a higher segregation stability [21], which can be further enhanced through the use of the p15A replicon instead of ColE1 replicon [22].

Design vector pACT7 that meet the specified conditions, were carried out in several stages.

A) Construction of intermediate plasmids pET21dRI.

C using the method of "inverse PCR and primers pETR1_F (SEQ ID No. 7) and pETR1_R (SEQ ID No. 8) on the matrix plaidy pET21d [21] using a set of Expand Long Template PCR system have a unique PCR fragment size of 5.4 TPN, which is isolated from the agarose gel as described in example 1. 100 ng of the obtained fragment hydrolyzing 5 units restrictase EcoRI (Fermentas) and are ligated using T4 DNA ligase. Received ligase mixture transform competent cells of Escherichia coli strain XL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F proAB lacIqZ□M15 Tn10 (Tetr)] (Stratagene, USA), then from the obtained ampicillin-resistant transformants isolated preparations of plasmid DNA using the set Wizard MiniPreps Kit (Promega, USA). Obtained DNA samples analyze joint hydrolysis of restrictase EcoRI/ > PST and BamHI/ > PST and select "positive" clones containing EcoRI/ > PST fragment sizes 4100 1300 and P.N. however due to the deletion of the BamHI site in BamHI/ > PST hydrolysate DNA plasmid CL is new there is a unique fragment 5400 BP, while in the drug pET21d plasmid DNA, hydrolyzed BamHI/ > PST, found fragments of size 1300 and 4100 P.N. Several positive clones was checked by sequencing using primers Trga (SEQ ID No. 9) and T7term (SEQ ID No. 10) and select the clone with modified area polylinker, not containing nonspecific mutations that indicate how pET21dR1.

B) Construction of plasmids pACYCpET.

To obtain the derived vector pET21dRI with the p15A replicon DNA plasmid pACYC177 [22] hydrolyzed together with restrictase BamHI+ > PST and was isolated from agarose gel, the fragment size 3000 BP, including the region of the replicon RA, the gene for resistance to kanamycin and C-terminal coding part of the gene beta-lactamase (resistance to ampicillin). Plasmid pET21dR1 hydrolyzed together BglII+ > PST and was isolated from an agarose gel slice 1.5 TPN, including the area of polylinker, the promoter and terminator T7-phage and N-terminal coding part of the gene beta-lactamase. Two fragments ligated received ligase mixture was used to transform E. Coli strain Xl1Blue and selected transformants resistant to both ampicillin and kanamycin.

Isolated from cultures of the obtained clones plasmid DNA was analyzed by restriction analysis with the endonucleases SmaI and selected clones, forming fragments with a size of 3.2 and 1.2 TPN One of the selected clones oboznachit is and how pACYCpET.

B) Constructing a vector pACT7

Removal of the gene beta-lactamase from the vector pACYCpET preparation of plasmid DNA hydrolyzing jointly by restrictase NaeI/FspI, isolated from the agarose gel, the fragment size 3,4 TPN, are ligated "himself" blunt ends" and ligase mixture transform competent cells of strain E.oli JM109. Selected kanamycin-resistant and ampicillin-sensitive transformants produce plasmid DNA and identify the correct clone according to restrictive analysis (the presence of fragments of size 1.2 and 2.2 TPN in SmaI-hydrolysates preparations of plasmid DNA).

Example 3. Construction of plasmids pSVH1811.

To create a plasmid capable of directing the synthesis BrdGL7ACA-acylase in E.coli cells were embedding the selected gene BrdGL7ACA-acylase under the control of the promoter of T7 phage vector pACT7.

The obtained PCR fragment of genomic DNA of strain Brevundiminas diminuta WCMW-1297 contains the complete coding sequence BrdGl7ACA-acylase and flanked by unique restriction sites EcoRI and SacI, missing in the coding sequence of the gene for subsequent directional embedding it under the control of T7 promoter created expression vectors.

Isolated from an agarose gel slice hydrolyzing restrictase EcoRI/SacI and clone into the EcoRI/SacI vector pACT7. Select kanamycin-resistant transformant the strain Escherichia coli JM109 according to the criterion of formation of fragments with a size of 2.2 and 3.4 TPN in EcoRI/SacI hydrolysates obtained preparations of plasmid DNA.

For selection of a clone carrying a box with intact genome BrdGL7ACA-acylase possible without mutations introduced through PCR amplification, check the ability of the selected plasmid to direct the synthesis of functionally active BrdGL7ACA-acylase when they transfer into the E.coli strain BL21(DE3).

For this individual transformants of the strain E. coli BL21(DE3)carrying the resulting plasmid, grown in LB-medium containing 30 μg/ml kanamycin, in an amount of 5 ml to a600of 1.0 OE. After this culture make IPTG to a final concentration of 1 μm and continue culturing for 18 hours at a temperature of 25-30°C. Cells are harvested by centrifugation and carry out the determination of the activity of Gl7ACA-acylase, which uses colorimetric method similar to the method proposed for the determination of 6-aminopenicillanic acid [24]. 100 μl of 65 mm solution glutaryl-7-ACA in 0.1M phosphate buffer (pH 7.0) is added to 900 μl of the cell suspension prepared in the same (0,1M phosphate) buffer containing 3 mg/ml of clavulanate potassium, and incubated the mixture at 37°C from 30 min to 4 hours. After a certain period of time the mixture is centrifuged, selected 500 μl of the supernatant and mix it with 3 ml of a mixture (2:1) 20% acetic acid and 0.5 M NaOH to stop the reaction. The sample add 0.5 ml of p-dimethylaminobenzaldehyde is Yes (PDAB) and after color development measure the absorbance at 415 nm. One unit of enzyme activity is accepted amount required to convert 1 μmol of substrate in these conditions for 1 min incubation.

Certain level so acylase activity in the analysed strains ranged from 1.0 u/ml to about 1.4 units/ml of culture.

Selected positive clones is sequenced and identify the clone carrying the box with the intact gene BrdGl7ACA-acylase, the coding sequence of which is identical to the sequence shown in the sequence listing under the number SEQ ID No. 5. The plasmids isolated from selected clones represent pSVH1811.

Example 4. Construction of expression vector of the hybrid protein BrdGl7ACA-cbd

A) Obtaining an intermediate plasmid pSVH0107.

To obtain a vector that allows the expression of the target enzyme in the form of a hybrid with the chitin-binding domain, first performed oligonucleotide-directed mutagenesis using a set of QuikChange® Site-Directed Mutagenesis Kit (Stratagene, USA) and primers Apa_up (SEQ ID No. 11) and Apa_dn (SEQ ID No. 12) with the purpose of introducing customers Vne1 in 551-554 position acylase gene (SEQ ID No. 5), being part of the plasmid vector pSVH1811.

After the reaction mixture was purified from impurities primers, using Wizard PCR preps Purification System (Promega, USA) and hydrolyzed in 5 units restrictase Dpn1 ((Fermentas). Received what MESU transformed competent cells of Escherichia coli strain XL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F proAB lacI qZ□M15 Tn10 (Tetr)] (Stratagene, USA). From the obtained kanamycin-resistant transformants were isolated preparations of plasmid DNA using a set of "Wizard MiniPreps Kit (Promega, USA). The resulting DNA samples were analyzed using PCR with primers 7_R and BRD2, for which the reaction mixture was separated in 1% agarose gel and revealed a single fragment 1260 BP PCR product restriction enzyme hydrolyzed in the Vne1 and selected positive clones, obnarujivaiu in an electrophoretic separation 2 fragment sizes 560 and 700 BP (clones that do not contain the correct site is detected only one fragment 1260 BP). Some of the positive clones were further verified by sequencing using primers 7__R and BRD2, the result of which was oatbran clone with Vne1 site in position 551-554 acylase gene (SEQ ID No. 5), not containing this non-specific mutations. Plasmid isolated from this clone was designated as pSVH0107.

B) the Allocation sequence that encodes a CBD domain of chitinase A1 B.circulans.

As the source of the gene CBD domain of chitinase A1 B.circulans used plasmid pTYB4 (New England Biolabs). Using primers BD_F (SEQ ID No. 13) and CBD_R (SEQ ID No. 14) on the matrix plaidy pTYB4 using Pfu polymerase was given a unique PCR fragment with a size of 0.2 TPN

The fragment was isolated from gel using the set Wizard PCR Preps Kit (Promega, USA) with the availa able scientific C with the instructions of the manufacturer and subjected to automated sequencing on the device ABIPrizm 3100 DNA Sequencer using primers BD_F and CBD_R and set Applera "fluorescent Big dye Cycle sequencing kit". The obtained sequence (SEQ ID No. 15) corresponds to the sequence that encodes a CBD domain of chitinase A1.

C) Obtaining plasmids pSVH0108.

Sequence encoding a hybrid protein BrdGl7ACA-cbd, obtained by combining contained in the vector pSVH0107 fragment encoding BrdGl7ACA and includes an additional site Vne1, and fragment encoding D-domain (see above).

To do this, 2 μg of plasmid DNA pSVH0107 hydrolyzed with the restriction enzyme Vne1, dephosphorylated using phosphatase, calf intestine to prevent closure of the vector to itself" and was isolated from agarose gel, the fragment size 5,6 TPN 5 ál of the mixture was used to transform competent cells of strain E.oli JM109 [19].

PCR fragment encoding CBD-domain, also hydrolyzed by restriction enzyme Vne1. Used the following criteria ligation: 1 ál vector (20 ng/μl), 1 μl of fragment (50 ng/μl), 2 μl 5 × ligase buffer (Gibco-BRL), 5 μl water and 1 μl T4 DNA ligase (1 u/ál, SibEnzyme). The mixture is incubated for 14 hours at 12°C, then warmed up for 15 min at 65°C, cooled in ice.

Received ligase mixture was used to transform competent cells of Escherichia coli strain XL1-Blue. From the obtained kanamycin-resistant transformants were isolated plasmid DNA and selected positive clones by PCR screening with primers BRD2 and CBD_F education PCR product is the size of 1000 BP

Several positive clones were additionally checked by sekvenirovanie and selected the clone with the insertion of CBD-encoding fragment that does not contain non-specific mutations. Derived from plasmid was designated as pSVH0108 (figure 2). This plasmid contained a sequence encoding a full hybrid protein BrdGl7ACA-cbd (SEQ ID No. 16), which represents the amino acid sequence of glutaryl acylase with the insertion of a CBD domain in the C-terminal region of alpha-subunit between amino acids R184 and T185.

Example 5. Obtaining recombinant strain-producer BrdGl7ACA-cbd.

The obtained recombinant plasmid pSVH0108 transformed strain E. coli BL21 (DE3) [21] [F-, ompT, hsdSB(rB-, mB-), dcm, gal (DE3)].

Since the primary transformants DE3 - recipients may differ significantly in the level of expression of a target protein [25], for the selection of the most active producer of protein BrdGl7ACA-cbd separate clones of the transformants were grown in pre-defined optimal for production Gl7ACA-acylase conditions: cells were introduced into 50 ml of LB medium, and cultivated at a temperature of 25°C to 1.0 SECOND, when a600after which was added inductor IPTG to a final concentration of 0.2 mm and continued incubation for 22 hours. Periodically, taking aliquots of the cell suspension grown individual transformants and used them the La define culture growth and activity of the enzyme. To obtain a rough extract the precipitated cells were destroyed by ultrasound with periodic cooling in ice (4×30 sec with an interval of 1 min). Cellular debris was removed by centrifugation. For determination of enzyme activity, which was conducted as described in example 3 was used received 50 ál of the clarified lysate.

Specific activity was expressed in units of enzyme activity (number of mcmole substrate per minute at 25°C) per mg of total protein in the clarified extract. The total protein content was determined by the method of Bradford [19].

On the basis of the obtained data was selected transformant No. 3, characterized best by the accumulation of biomass and higher yield functionally active enzyme (table 1). He was designated BL21 (DE3)/pSVH0108 and used in further work to accumulate culture and preparation of recombinant strain.

Table 1
Growth parameters of culture and activity BrdGl7ACA-cbd from individual transformants of the strain E. coli BL21 (DE3) with plasmid pSVH0108
Individual clonesGrowth
culture, A600
Activity BrdGl7ACA-cbd, IU/mg proteinOutput BrdGl7ACA-cbd, IU/ml
No. 15,080001200
No. 24,565001050
No. 35,2100001400
No. 44,370001100
No. 55,163001000

Example 6. Partial purification of the hybrid protein BrdGl7ACA-cbd and its immobilization on chitin sorbent.

Sufficiently high levels of expression variation glutaryl-acylase obtained in the strain to a certain extent simplified the task of developing methods for partial purification of the hybrid protein, which was carried out as described below. The resulting biomass recombinant strain (5 g) is suspended in 20 ml buffer A (100 mm sodium phosphate, pH 8.0, 5 mm 2-mercaptoethanol, 10 mm EDTA, 0.3% setitimer ammonium bromide (BECOMING), 2% glycerol) and was destroyed by ultrasonic disintegration. The supernatant was collected debris after washing with the same buffer is discarded. Ballast proteins from the supernatant precipitated with ammonium sulfate at 35% saturation, R is combinatul BrdGl7ACA-cbd was obtained as a precipitate when saturated with ammonium sulfate to 60%. The precipitate was dissolved in 10 ml of buffer B (10 mm sodium pyrophosphate, pH 8.0, 5 mm 2-mercaptoethanol, 2 mm EDTA, 10% glycerol, 300 mm NaCl) and applied to a column of chitin pellets (2×5 cm). The column was washed with buffer B until the disappearance of the absorption at A, selected the sample of sorbent and used to determine the acylase activity and total protein. The stages of purification and immobilization are shown in Table 2. The electrophoresis method, we established a high purity recombinant acylase, consisting of two subunits: beta 54 kDa and alpha+CBD - 20 kDa.

The data obtained allow us to conclude that the established structures of the chimeric protein is fully retains the properties of the two polypeptide components.

Table 2
Purification and immobilization BrdGl7ACA-cbd
Cleanup stepTotal protein (mg)The oxidase activity (µmol/min)Output %The degree of purification (fold)
General (IU)Unit (IU/mg)
Cell-free extract350of 550,00016001001
the residue (60%)
Immobilization of chitin sorbent3535000010000646
Note: experience has taken 5 g wet biomass E.coli

Example 7. Characterization of the recombinant strain E. coli BL21(DE3)/ pSVH0108.

Cells obtained recombinant Escherichia coli strain BL21(DE3)/ pSVH0108 characterized by the following features.

Morphological features of the Cells have an elongated rod-like shape, when the division is not packouts.

Cultural characteristics

Cells grow well on commonly used nutrient media. The generation time of about 30 min in liquid LB-medium. 2-2,5% nutrient agar "Difco" formed round, smooth, yellowish colonies with smooth edges. When grown in liquid LB - and YT-media forms of intensive ditch the traveler turbidity.

Physiological and biochemical characteristics

The optimal cultivation temperature from 25 to 30°C, the optimum pH of 7.6. Source of nitrogen are organic compounds (in the form of tryptone, yeast extract).

The level of synthesis BrdGl7ACA-cbd in the engineered strain is about 100 mg/l when the titer of the culture of 1×109cells/ml, which follows from the definition data GL7ACA-acylase in the samples of biomass producer strain by using a specific enzymatic reaction. The value of the specific activity of the hybrid protein BrdGl7ACA-cbd is about 10,000 IU/mg protein (table 2) and close to the number of the specific activity of purified recombinant "unmodified" Gl7ACA-acilis described previously [9-12, 15].

The list of cited sources

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2. Arroyo, M., de, 1.M., I, Acebal, C., & Castillon, M.P. Biotechnological applications of penicillin acylases: state-of-the-art. Appl. Environ. Biotechnol. 60, 507-514 (2003).

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6. Parmar, A., Kumar, H., Marwaha, S.S., & Kennedy, J.F. Recent Trends in Enzymatc Conversion of Cephalosporin C to 7-Aminocephalosporanic Acid (7-ACA). Critical Reviews in Biotechnology 18, 1-12 (1998).

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16. Lee, Y.S., Kim, H.W., Lee, K.B., & Park, S.S. Involvement of arginine and tryptophan residues in catalytic activity of glutaryl 7-aminocephalosporanic acid acylase from Pseudomonas sp.strain GK16. Biochim. Biophys. Acta 1523, 123-127 (2000).

17. Kim, Y. & Hol, W.G. Structure of cephalosporin acylase in complex with glutaryl-7-aminocephalosporanic acid and glutarate: insight into the basis of its substrate specificity. Che.Biol. 8, 1253-1264 (2001).

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1. Recombinant DNA which encodes a functionally active hybrid protein (BrdG17ACA-cbd), consisting of the amino acid sequence of the acylase glutaryl-7-is aminocephalosporanic acid strain Brevundimonas diminuta BKM-1297 and chitin-binding domain of chitinase Al Bacillus circulans, and characterized by nucleotide sequence SEQ ID No. 16.

2. Recombinant plasmid pSVH0108 for synthesis of a hybrid protein BrdG17ACA-cbd in E.coli cells, which are formed by the vector rst consisting of a fragment of the modified area polylinker plasmid pET21d containing the promoter and terminator of RNA polymerase of phage T7; divided by the area of polylinker, and fragment of plasmid pACYC177, containing the replicon RA and the gene for resistance to kanamycin, interconnected as shown in figure 2, and the sequence of the recombinant DNA according to claim 1, integrated in polyglycerol the region of the specified vector.

3. The recombinant strain E. coli BL21(DE3)/pSVH0108 cbd producing a hybrid protein BrdG17ACA-cbd.


Same patents:

FIELD: medicine.

SUBSTANCE: invention can be used in producing vaccines against Streptococcus agalactiae - a representative of streptococci group B (SGB) in diagnostics of the diseases - for creation of a detection system of immunoglobulin A level in biological fluids, in immunochemistry as accessible immunochemical reagents (affine recovered IgA fragments). Offered unique recombinant DNA are produced by polymerase chain reaction (PCR) with using chromosomal DNA of strain 219/4849 Ibc of serotype SGB and unique primers. One recombinant DNA contains three nucleotide substitutes in comparison with an initial site of chromosomal DNA. The following cloning of amplified fragments is carried out in a linear vector pGEM-T Easy, and at the final stage by the system of express ionic vectors pQE30/31/32 in E coli JM 109. The produced recombinant DNA code amino acid sequences of recombinant polypeptides exhibiting ability to connect selectively various molecular forms of IgA and designated as P6, P7, P8. Polypeptide P6 causes synthesis of long circulating high-affine anti-Rb antibodies possessing protective properties against SGB.

EFFECT: application of the invention provides production of recombinant polypeptides based N-terminal conservative part of surface Bac SGB of Ibc serotype and containing a first IgA-connecting site A with changed or native sequence MLKKIE, polypeptide exhibits immunologically relevant and protective properties, and they also high selectively connect IgA.

12 cl, 20 dwg, 4 tbl, 19 ex

Expressing system // 2385348

FIELD: medicine.

SUBSTANCE: invention concerns the method for making an immunogenic reagent which causes immune response on infection Bacillus anthracis, including one to several polypeptides which together represent three domains of a full-size protective antigen (PA) from B anthracis or their versions, and at least, one of specified domains contains domain 1 or domain 4 of the PA, or its version. Said polypeptides of specified immunogenic reagent, and the full-size PA are produced as a result of expression in a recombinant cell E.coli. The invention also discloses an expression vector and nucleic acid with percent of residual guanidine and cytosine more than 35%, coding immunigenic polypeptide which is said protective antigen (PA).

EFFECT: high-yield immunogenic polypeptide.

13 cl, 5 dwg, 3 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: there is designed recombinant plasmid DNA pYfi-gfp enabling production of chimeric fluorescent protein GFPaav with five modified amino acids, corresponding to the first five amino acids of protein YfiA with controlling a promotor of gene YfiA in presence of toxic agents. Plasmid has size 3730 base pairs. Recombinant strain Escherichia coli JM109-pYfi contains a genetic design under given invention and produces fluorescent protein GFPaav in presence of toxic agents damaging a cell.

EFFECT: invention allows registering the presence of toxic agents in a medium with increasing the fluorescence level of cells of produced strain.

2 cl, 4 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: there is designed recombinant plasmid DNA pSC13D6 containing a gene of one-chained antibody to tick-borne encephalitis virus sc13D6. Plasmid consists of 3782 basepairs and contains: a plasmid vector pGEMl, phage T7 promotor and unique restriction sites. There is also discovered strain of bacteria Escherichia coli BL21 (DE3)/pSC13D6 - a producer of a virus neutralising one-chained antibody sc13D6 to tick-borne encephalitis virus.

EFFECT: invention allows producing one-chained antibodies sc13D6 to tick-borne encephalitis virus able to inhibit it effectively.

2 cl, 5 dwg, 5 ex

FIELD: chemistry, medicine.

SUBSTANCE: novel antibodies and fragments of human antibodies are bound with GDF-8 in a specific way and inhibit its activity in vitro and/or in vivo. On the basis of said invention pharmaceutical composition is created, which can be used for diagnostics, prevention or treatment of degenerative dysfunctions of muscle or bone or disorders of insulin metabolism.

EFFECT: extending range of arsenal of technical means used in treatment of diseases related to muscular, bone tissue or insulin metabolism.

FIELD: medicine.

SUBSTANCE: there are offered recombinant protein molecule M2E-HBC, and virus-like particles formed of such molecules. The recombinant virus-like particle based on nuclear antigen of hepatitis B virus represents surface polypeptides of outer domain M2 of avian influenza virus protein. The produced virus-like particles are characterised with high immunogenicity. There is also disclosed vaccine for the infection caused by avian influenza virus, including such virus-like particles as an active agent.

EFFECT: preparations are active to various strains of avian influenza virus and can be considered as a candidate for universal avian influenza virus type A vaccine.

7 cl, 8 dwg, 4 tbl, 8 ex

FIELD: biology.

SUBSTANCE: present invention relates to biotechnology and genetic engineering. Proposed here is a set of lux-biosensors, consisting of Escherichia coli cells obtained using recombinant DNA technology, containing plasmids with bacterial luxCDABE genes controlled by stress inducible promoters PgpoE and PgrpE and non-inducible Plac promoter.

EFFECT: invention can be used in evaluating degree of contamination of the environment by hydrophobic detergents.

FIELD: medicine.

SUBSTANCE: hybrid protein - human insulin precursor consists of N-end fragment of human gamma-interferon connected through peptide linker with amino acid sequence of human proinsulin. Recombinant human insulin is obtained by cultivation of Escherichia coli JM109/pHINS11 strain-producer, carrying plasmid pHINS11, isolation of inclusion bodies and their dissolving in buffer which contains urea and dithiotreitole. Then hybrid protein re-naturation, sedimentation of admixture compounds, purification of re-naturated hybrid protein by ion-exchanging chromatography, combined fermentative hydrolysis of hybrid protein with tripsin and carbopeptidase B are carried out. At the last stage insulin purification with cation-exchanging chromatography and method of highly efficient reverse phase liquid chromatography are carried out.

EFFECT: simplification of obtaining highly purified recombinant human insulin and increase of its output.

6 cl, 1 dwg, 4 tbl, 5 ex

FIELD: chemistry, pharmacology.

SUBSTANCE: plasmid pAS-2 is formed by HindIII/BamHI-fragment of plasmid pPINS07 incorporating a part of artificial gene encoding lgG-binding protein domain A from S. aureus and peptide His6GlySerArg, and HindIII/BamHI-fragment encoding proinsulin Aspart. Plasmid pAS-2 ensures in cells E. coli the biosynthesis of hybrid protein wherein B domain lineage of staphylococcal protein A Staphylococcus aureus is linked via peptide linker His6GlySerArg with amino acid sequence of human being proinsulin Aspart. The human being strain of bacteria Escherichia coli BAS2 produced by transforming the cells of the strain by plasmid pAS-2 produces the said hybrid protein.

EFFECT: production of human being insulin Aspart by simplified process with high yield.

2 cl, 4 dwg, 4 ex

FIELD: chemistry, genetics.

SUBSTANCE: invention relates to the field of genetic engineering and can be used for obtaining human interleukin-13 (IL-13). On the basis of plasmid pTrcTEGF and optimised for bacterial expression cDNA encoding a mature form of human interleukin-13 a recombinant plasmid DNA pTrcTREN-IL13 is constructed. This plasmid DNA provides biosynthesis of polypeptide with properties of human interleukin-13 in cells of E.coli. As a result of transformation of strain of bacteria Escherichia coli BL21(DE3) by the said plasmid, strain of E.coli BL21(DE3)/pTrcTREN-IL13/pTrcTREN-IL13 is obtained, which produces polypeptide with properties of human interleukin-13.

EFFECT: ensuring an increase in the level of heterological expression of polypeptide with properties of human interleukin-13 at reduced quantity of inductor.

2 cl, 4 dwg, 1 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: invention represents a combination containing VEGF Trap and 5-fluorouracil to be applied in treatment of neoplasms.

EFFECT: higher effectiveness of the combination ensured by therapeutic synergism of its components, and reduced toxicity.

4 cl, 1 ex, 1 tbl

FIELD: biotechnologies.

SUBSTANCE: plants are transformed with application of nucleic-acid constructs, which contain the first nucleotide sequence that codes γ-sein, or its fragment, which is able to direct and retain protein in endoplasmic reticulum of plant cell, the second sequence of nucleic acid, which codes aminoacid sequence, which is specifically split by ferment or chemical compounds, and the third sequence of nucleic acid, which codes target peptide or protein.

EFFECT: transformation of plant by such constructs makes it possible to produce fused proteins accumulated in endoplasmic reticulum of cells in the form of protein bodies, from which target proteins may be extracted, in particular calcitonin.

47 cl, 19 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: there is offered molecule of nucleic acid inducing CEA immune response, containing a nucleotide sequence that codes a fused protein on a basis of carcinoembryonal antigen (CEA) or its functional version fused with a subunit B of thermolabile enterotoxin E coli. There are described versions thereof, as well as the related purified protein. There is disclosed an expression vector containing said molecule of nucleic acid, and a host-cell containing specified vector. There are described adenoviral vaccinal vector for inducing the immune response and a vaccinal plasmid on the basis of the specified molecule.

EFFECT: application of the invention allows to inducing the immune response in a mammal which is stronger, than that induced with natural CEA that can find application in medicine for cancer treatment.

20 cl, 62 dwg, 20 ex

FIELD: medicine.

SUBSTANCE: there is prepared identification peptide with amino acid sequence: Gly-Pro-Ala-Pro-Gln-Pro-Asp-Glu-Asp-Leu-Lys-Arg-Gln. The prepared peptide is applied to identify, purify and recover the recombinant proteins containing it. For making the recombinant proteins with a polypeptide label, an expression vector pDED is applied. Said vector contains a nucleotide sequence coding amino acid sequence of the identification protein.

EFFECT: invention allows extending range of the methods to identify the recombinant proteins.

14 cl, 6 dwg, 1 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: invention refers to genetic engineering and can be used in medicine. The mucosal vaccine contains effective amount of hybrid protein consisting of oncoprotein E7 of human papilloma virus fused with heat-shock protein of mycobacteria Hsp70, chitosan related to hybrid protein 1:0.1-10 and additives pharmaceutically acceptable manufacturing of suppositories. The mucosal vaccine is used in therapy of the diseases associated with human papilloma virus.

EFFECT: possibility for multiple improvement of clinical effectiveness of diseases associated with human papilloma virus, considerable reduction of treatment cost in comparison with common techniques of treating cervical carcinoma and "РПК"; elimination of injection by-effects undesirable and extremely dangerous for the patent's life, eg anaphylactic shock, owing to local application; simplification of medical process - the patient can receive medical treatment out of clinic by independent introduction of the preparation.

6 cl, 4 dwg, 2 tbl, 10 ex

FIELD: medicine.

SUBSTANCE: invention is related to preparation of protein, binding tumour necrosis factor (TNF), and may be used in medicine. Strain-producer of baculovirus BvG2RIgG is created with the help of recombinant plasmid DNA pFastBac-G2R-IgG with size of 6444 p.n. and molecular mass 4.18 mDa, which bears fragment of smallpox virus genome of strain India-1967, which codes protein that binds TNF, and fragment of human genome, which codes fragment of heavy chain of human antibody G. Produced strain produces soluble chimeric protein, which consists of smallpoz virus protein, which binds TNF, and fragment of heavy chain of human antibody G.

EFFECT: wider spectrum of new generation preparations intended for treatment of human diseases related to hyperproduction of tumour necrosis factor.

2 cl, 3 dwg, 1 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention is related to nucleic acids and multidomain proteins, which are able to bind vessel endotheliocyte growth factor (VEGF), and may be used in medicine. Recombinant method is used to produce polypeptide, which consists of component (R1R2)X and, unnecessarily, multidomain component (MC), which represents aminoacid sequence with length from 1 to 200 of amino acids, having at least one remainder of cysteine, where X≥1, R1 means antibody-like (Ig) domain 2 of VEGF receptor Llt-1, and R2 means Ig-domain 3 of VEGF receptor Flk-1. Produced fused polypeptide does not contain multidomain component in case, when X=2, and in case when X=1, multidomain component represents aminoacid sequence with length from 1 to 15 amino acids. Produced polypeptide is used in composition of pharmaceutical compound for VEGF-mediated disease or condition.

EFFECT: invention makes it possible to produce highly efficient trap of VEGF, special structure of which is suitable for local introduction into specific organs, tissues or cells.

16 cl, 3 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and can be used for screening of compounds with properties of agonists or antagonists of leptin receptor. There is disclosed method for detecting leptin receptor definition that provides contact of a candidate compound and cells expressing after contransfection with two expressing vectors, respectively, two fused proteins, one of which consists of a short isoform of leptin receptor (OBRs) and an energy donor protein (preferentially luciferase), and the second - of OBRs and an energy acceptor protein (preferentially GFP or its mutant form); measurement of energy transfer (BRET) between the fused proteins; comparison of its value to the relevant indicator measured in the same system, however without tested compound, and estimated result where higher energy transfer indicates binding of the candidate compound-candidate with leptin receptor. Prospective application of the invention is related to development of the preparations for prevention or treatment of disease wherein leptin or its receptor are involved.

EFFECT: development of effective method for detecting leptin receptor ligands.

4 cl, 15 dwg, 3 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, specifically to a method of producing recombinant protein human albumin-interleukin-2 or recombinant protein human albumin-alpha 16-interferon, modified by attachment of human albumin. The method involves technology of culturing yeast strain Pichia pastoris PS106/pPIC9HAbIL-2 or yeast strain Pichia pastoris PS106/pPIC9HAbIFNa-16 in modified culture medium BMGY, after which induction synthesis of target proteins is carried out at low temperature. Further, cells are removed and the medium is concentrated. Target proteins are then precipitated using ammonium sulphate or polyethyleneglycol 3350. Target proteins are then separated by gel filtration on Sephacryl HR 200 or BioRad P-300 sorbents. Finally, affinity chromatography is then done on Cibacron F3GA sorbent.

EFFECT: invention simplifies and increases efficiency of the technology of purifying target proteins, and also allows for obtaining biologically active hybrid proteins, suitable for making medicinal agents.

3 cl, 1 tbl, 5 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, specifically to a method of identifying γ-secretase and its inhibitors and can be used in medicine when searching for active compounds for treating Alzheimer's disease. A genetic structure is formed, which codes fused protein, which contains a signal peptide and amino acid sequence GAIIGLMVGGVVIATVIVITLVML. In the obtained fused protein, except the GAIIGLMVGGVVIATVIVITLVML sequence, all sites acting as a signal for endo- or exocytosis, and/or protease splitting site are excluded.

EFFECT: invention allows for highly effective identification of γ-secretase or substances which inhibit its activity by reducing background signal and increasing specificity of the signal.

41 cl, 4 dwg, 17 ex

FIELD: biotechnologies.

SUBSTANCE: invention represents polypeptide, having α-L-arabinofuranosidase activity selected from the following polypeptides: polypeptide with SEQ ID No. 2, polypeptide, amino-acid sequence of which is located between positions 28 and 400 SEQ ID No. 2, fragment of polypeptide with SEQ ID No. 2, having activity of α-L-arabinofuranosidase, polypeptide having activity of α-L-arabinofuranosidase B and expressing 80% identity with polypeptide SEQ ID No. 2. Invention also relates to polynucleotide, which codes this polypeptide, expression cassette and vector, containing polynucleotide, and master organism that contains this polypeptide.

EFFECT: expanded arsenal of mediums for hydrolysis of α-L-arabinofuranosyl links in arabinofuranosyl-oligosaccharide compounds.

9 cl, 6 dwg, 2 tbl, 1 ex