Modified colony-stimulating factor granulocyte person and the method of its production, dna expressing vector

 

The invention relates to biotechnology, in particular genetic engineering, and can be used to produce secreted modified colony-stimulating factor granulocyte person (hG-CSF). Modified hG-CSF produced by substitutions in the amino acid sequence of hG-CSF wild type 1st amino acid to Ser or 1-Oh and 17-Oh - Ser, 2nd amino acids at the Met and the 3rd on Val, or 2nd at the Met, 3rd on Val and 17th on Ser, or 17th on Thr. The cells of the microorganism, transformed by a vector containing a DNA encoding the modified hG-CSF, cultivated with receipt and secretion in periplasm hG-CSF. The invention allows to effectively Express and secrete the microorganism hG-CSF, preserving the biological activity of the protein of the wild type. 4 C. and 10 C.p. f-crystals, 11 tab., 1 PL.

The technical field,

The present invention relates to a modified colony-stimulating factor granulocyte person (hG-CSF), gene, codereuse the specified peptide vector containing the specified gene, a microorganism transformed by the specified vector, and the method of obtaining modified hG-CSF using the specified microorganism.

Background of the invention

Colonystimulating factor of macrophages (M-CSF) and colony-stimulating factor granulocyte (G-CSF), produced by T-cells, macrophages, fibroblasts and endothelial cells. GM-CSF stimulates the stem cells of granulocytes or macrophages, inducing their differentiation and proliferation of colonies of granulocytes or macrophages. M-CSF and G-CSF induce, respectively, the formation of colonies of macrophages and granulocytes. In vivo G-CSF induces the differentiation of cells of the bone marrow and enhances the function of Mature granulocytes and, therefore, determine their clinical significance in the treatment of leukemia.

G-CSF human (hG-CSF) is a protein consisting of 174 or 177 amino acids, and the variety including 174 amino acids, has a higher activity, stimulating neutrophils (Morishita, K. et al., J. Biol. Chem., 262, 15208-15213 (1987)). Amino acid sequence of hG-CSF, consisting of 174 amino acids, shown in Fig.1. Conducted multiple studies on mass production of hG-CSF with the help of the gene encoding specified hG-CSF.

For example, the company Chugai Pharmaceuticals Co., Ltd (Japan) was presented to the amino acid sequence of hG-CSF and its encoding gene (Korean patent publication No. 91-5624 and 92-2312) and described the method of obtaining a protein having the activity of hG-CSF, a method of genetic recombination (peyrovani hG-CSF, using genomic DNA or cDNA, containing polynucleotide encoding hG-CSF. Glycosylated hG-CSF has a chain of O-glycosidic sugar, but it is known that the chain is not necessary to maintain the activity of hG-CSF (Lawrence, M. et al., Science, 232, 61 (1986). In addition, it is well known that the production of glycosylated hG-CSF with the use of mammalian cells requires the use of expensive materials and devices, so this method is unacceptable from an economic point of view.

Attempts were made to produce non-hG-CSF using microorganisms, such as E. coli. As a result of performing such studies are the hG-CSF containing 175 or 178 amino acids methionine residue attached at the N-Terminus, due to the use of the microorganism of the initiating ATG codon. However, additional methionine residue causes an undesirable immune response in humans with the introduction of recombinant hG-CSF (European patent publication No. 256843). In addition, most methioninamide hG-CSF produced in E. coli, is deposited in the cells as insoluble Taurus inclusion that need to be converted into active form by re-laying circuit when significant the CSO type, involved in the formation of disulfide bonds, while the last remnant of the causes aggregation of the product hG-CSF in the process of re-laying circuit that reduces the product yield.

To solve the problems associated with the production of a foreign protein in a microbial cell, there have recently been attempts to create a method based on the effective secretion of the target protein through the membrane of the bacterial cell in the extracellular domain.

For example, when implementing the method using the signal peptide required protein expressed in the form of a fused protein in which the signal peptide attached to the N-end of the protein. When passing through the slit protein through the cell membrane signal peptide is removed by the enzyme and the desired protein is secreted in Mature form. The way secretory production is effective, as produced amino acid sequence is usually identical to the sequence of the wild type. However, the product yield in the process secretory production is often quite low due to insufficient transfer through the membrane and subsequent treatment. This is consistent with the known data that the yield of protein of a mammal, the products have not been considered suitable for efficient expression and secretion of soluble hG-CSF, not having an additional methionine residue attached to the N-end.

The authors of the present invention previously reported on the use of new secretory signal peptide, obtained by modifying the signal peptide thermoresistances enterotoxin II E. coli (open Korean patent publication No. 2000-19788), when the production of hG-CSF. In particular, was received expressing a vector containing a gene hG-CSF attached to the 3'-end of the modified signal peptide thermoresistances enterotoxin II E. coli, and expressed biologically active, Mature hG-CSF with the use of E. coii, transformed above expressing vector. However, most expressed hG-CSF accumulates in the cytoplasm and not in periplasm.

The authors of the present invention attempted to create an efficient secretory way of getting hG-CSF in the microorganism and found that the modified hG-CSF obtained by replacing at least one amino acid residue, in particular 17-th residue of cysteine, hG-CSF wild type another amino acid that helps to maintain the biological activity of the protein of the wild type, while in the microorganism can be effectively Express and the NSS signal peptide.

A brief statement of the substance of the invention

The aim of the present invention to provide a modified colony-stimulating factor granulocyte person (hG-CSF), which can efficiently be produced by using a microorganism.

Another objective of the present invention to provide a gene encoding the indicated peptide, and the vector containing the specified gene

Another objective of the present invention to provide a microorganism transformed by the specified vector.

Another objective of the present invention is the representation of a method of obtaining hG-CSF without methionine residue attached to aminobenzo, using the specified microorganism.

One object of the present invention is modified hG-CSF, characterized in that at least one of the 1st, 2nd, 3rd and 17th amino acids of hG-CSF wild type has been replaced with another amino acid.

Brief description of drawings

The above and other objects and features of the present invention will be apparent from the following description of the invention with reference to the accompanying drawings, where:

In Fig.1 shows the nucleotide and amino acid sequences colony-stimulating factor granulocyte the Oia vector pT-CSF.

In Fig.3 shows a method of creating a vector pT14S1SG.

In Fig.4 shows a method of creating a vector pT14SS1SG.

In Fig.5 shows a method of creating a vector pT140SSG-4T22Q.

In Fig.6 shows a method of creating a vector pT14SS1S17SEG.

In Fig.7 shows a method of creating a vector pTO1SG.

In Fig.8 shows a method of creating a vector pBADG.

In Fig.9 shows a method of creating a vector pBAD2M3VG.

In Fig.10A and 10b shows the results of analyses by the method of Western blotting, confirming the expression of hG-CSF and different modified hG-CSF in recombinant cell lines, and indicated the molecular weight of expressed proteins.

In Fig.11 shows the cellular activity of hG-CSF and the modified hG-CSF produced in recombinant cell lines.

Detailed description of the invention

Modified hG-CSF of the present invention receives, replacing one or more amino acids of hG-CSF wild type (SEQ ID NO: 2), preferably the 1st, 2nd, 3rd and 17 amino acids by other amino acids. More preferred factors receive, replacing the 17-th amino acid of hG-CSF amino acid, which is not charged at neutral pH. Specific examples of preferred modified hG-CSF have the amino acid sequence of hG-CSF wild type for escluse X;

(c) the 2nd amino acid is Met and 3rd amino acid is Val;

(d) the 2nd amino acid is Met, the 3rd amino acid is Val and 17th amino acid is X; or

(f) the 17th amino acid is X,

where x is the amino acid that is not charged at neutral pH, preferably Ser, Thr, Ala or Gly, more preferably Ser.

Four of the five residues s in hG-CSF are involved in the formation of disulfide bonds, while the 17th Cys residue remains unbound in its natural state. When recombinant cells expressed a large number of hG-CSF, 17th Cys residue involved in the formation of intermolecular disulfide bonds, resulting in an accumulation of agglomerated hG-CSF in the cytoplasm. However, in a modified hG-CSF of the present invention, having in position 17 not s, and the other amino acid residue, such a problem does not exist, so it can effectively produce secretory way, using properly transformed microorganism.

Modified hG-CSF of the present invention may be encoded gene containing a nucleotide sequence derived from the amino acid sequence of the modified hG-CSF in accordance with Geneva the degeneracy of the codon therefore, the scope of the present invention includes all of the nucleotide sequence derived from the amino acid sequence of the modified hG-CSF. Preferably the sequence of the gene modified hG-CSF includes one or more preferred E. coli codons.

Thus obtained gene can be inserted into conventional vectors to get expressing vector, which, in turn, can be an acceptable host, such as E. coli. Expressing the vector may further comprise a signal peptide. A typical signal peptide is the signal peptide thermoresistances enterotoxin II E. coli (SEQ ID NO: 53), the modified signal peptide thermoresistances enterotoxin II E. coli (SEQ ID NO: 54), signal peptide beta-lactamase (SEQ ID NO: 24), the signal peptide Gene III (SEQ ID NO: 42) or a modified peptide, but these peptides do not limit the number of signal peptides suitable for use in the present invention. The promoter used to obtain expressing vector of the present invention, is of a type that is able to Express the heterologous protein in a microorganism host. In particular, lac, Tac and arabinosyl the promoter are the invention, used as example, are pT14SS1SG, PT14SS1S17SEG, pTO1SG, pTO1S17SG, pT17SG, pT17TG, pT17AG, pT17GG, pBAD2M3VG, pBAD17SG and pBAD2M3V17SG.

Expressing the vectors of the present invention can be introduced in such microorganisms, such as E. coli BL21(DE3) (Novagen), E. coli XL-1 blue (Novagen), by the usual transformation (Sambrook et al., see above), while receiving the transformed E. coli BL21(DE3)/DT14SS1SG (NM 10310), E. coli BL21(DE3)/pT14SS1S17SEG (HM 10311), E. coli BL21(DE3)/ pTO1SG (HM 10409), E. coli BL21(DE3)/pT1S17SG (HM 10410), E. coli BL21(DE3)/pT17SG (HM 10411), E. coli BL21(DE3)/pT17TG (HM 10413), E. coli BL21 (DE3)/pT17AG (HM 10414), E. coli BL21(DE3)/pTO17GG (HM 10415), E. coli BL21 (DE3)/ pBAD2M3VG (HM 10510), E. coli BL21 (DE3)/ pBAD17SG (HM 10511) and E. coli BL21 (DE3)/ pBAD2M3V17SG (HM 10512).

Among the transformed microorganisms are preferred transformants of E. coli BL21(DE3)/ pT14SS1S17SEG (HM 10311), E. coli BL21(DE3)/ pT1S17SG (HM 10410), E. coli BL21(DE3)/ pT17SG (HM 10411) and E. coli BL21 (DE3)/ pBAD2M3VG (HM 10510), which were deposited in the Korean Central register of cultures of microorganisms (cssm) (address: Department of Food Engineering, College of Eng., Yonsei University, Sodaemungu, Seoul 120-749, Republic of Korea), March 24, 1999, under access numbers xsm-10154, xsm-10151, xsm-10152 and xsm-10153 in accordance with the provisions of the Budapest Treaty on the international recognition of registration of microorganisms during the patent examination.

The modified protein hG-CSF truly izaberete fitsirovannye protein hG-CSF, secretion of the modified protein hG-CSF in periplasm and excretion of the modified protein hG-CSF from periplasm.

The transformed microorganism can be cultivated in accordance with the known method (Sambrook et al., see above). Culture of microorganisms centrifuged or filtered, collecting thus the microorganism secreting modified protein hG-CSF. The transformed microorganism can be destroyed in the usual way (Ausubel, F. M. et al., Current Protocols in Molecular Biology, (1989)) to obtain the solution periplasm. For example, the microorganism can be destroyed in a hypotonic solution, such as distilled water, using osmotic shock. Modified hG-CSF can be isolated from solution periplasm conventional method (Sambrook et al., see above), for example, by using ion-exchange chromatography, gel-filtration chromatography on columns or immunogen-chromatography on columns. For example, hG-CSF can be cleaned by sequentially performing chromatography on a column of CM-separate and chromatography on columns with phenyltetrazol.

The modified protein hG-CSF, obtained according to the present invention, does not have a methionine residue at the N-Terminus and has a biological activity, which is equal to or higher than the activity h are given to more fully illustrate the present invention and do not limit its scope.

Example 1. Obtaining the gene encoding hG-CSF cDNA encoding hG-CSF, get, performing polymerase chain reaction (PCR) using matrix hG-CSF (R&D system, USA). Used starters are described in U.S. patent NO: 4810643.

To obtain gene cDNA encoding the Mature hG-CSF, vector pUC19-G-CSF (Biolabs, USA) and subjected to PCR using a dose of SEQ ID NOS: 3 and 4. The seed SEQ ID NO: 3 is used to obtain the restriction site NdeI (5'-STATS-3') upstream of the codon for the first amino acid (threonine) of the Mature hG-CSF, and the seed SEQ ID NO: 4 is used to obtain the restriction site BamHI (5'-GGATCC-3') below the termination codon.

Amplificatory gene hG-CSF digested using NdeI and BamHI and receive the gene encoding the Mature hG-CSF. Gene hG-CSF is administered at the site of NdeI/BamHI vector pET14b (Novagen, USA), while receiving the vector pT-CSF.

In Fig.2 shows the above-described method of creating a vector pT-CSF.

Example 2. Create a vector containing the gene encoding the signal peptide of enterotoxin E. coli and modified hG-CSF

(Stage 1) Cloning of the gene signal peptide of enterotoxin II E. coli

To obtain gene signal peptide of enterotoxin II E. coli, create a pair of complementary oligonucleotides having SEQ ID NOS: 5 and 6, on the basis of the nucleotide sequence of>/p>The above oligonucleotides designed for BspHI restriction site (sites that are complementary to the restriction sites NcoI) above from the initiating codon of enterotoxin II E. coli and MluI restriction site introduced by silent substitutions at the other end.

Both of the oligonucleotide is annealed at 95°C, thus obtaining the DNA fragments with "blunt" ends, having nucleotide sequence encoding a signal peptide of enterotoxin II E. coli (gene STII).

Gene STII enter the SmaI site of the vector pUC19 (Biolabs, USA) and get the vector pUC19ST.

(Stage 2) Obtaining the gene encoding STII/hG-CSF

To obtain the gene encoding STII/hG-CSF, vector RT-CSF obtained in preparative example 1 was subjected to PCR using a dose of SEQ ID NOS: 7 and 8. The seed SEQ ID NO: 7 is designed for replacing the first codon hG-CSF by the Ser codon, and the seed SEQ ID NO: 8 is used to obtain the restriction site BamHI (5'-GGATCC-3') below the termination codon.

Amplificatoare DNA fragments were cleaved using MluI and BamHI and then injected at the site MluI/BamHI vector pUC19ST obtained in stage 1, and the receive vector PUC19S1SG. The thus created vector pUC19S1SG contains the gene encoding STII/hG-CSF (referred to as genome STII-hG-CSF).

Vector pUC19S1SG disintegrated with BspHI and BamHI while 1SG.

In Fig.3 shows the above-described method of creating a vector PT14S1SG.

(Stage 3) Join sequences Shine-Dalgarno enterotoxin II E. coli to gene STII-hG-CSF

Vector pT14S1SG obtained in stage 2, is subjected to PCR using a dose of SEQ ID NOS: 9 and 10. The seed SEQ ID NO: 9 is used to obtain the sequence of the Shine-Dalgarno (called sequence STII SD) enterotoxin II E. coli and restriction site XbaI, and the seed SEQ ID NO: 10 is used to obtain the restriction site BamHI below the termination codon of the Mature hG-CSF with the purpose of creating a DNA fragment (STII SD-STII-hCSF), containing the gene STII SD and STII-hG-CSF.

Fragment STII SD-STII-hG-CSF digested using XbaI and BamHI and then injected at the site XbaI/BamHI vector pET14b (Novagen, USA), obtaining the vector pT14SS1SG.

In Fig.4 shows the above-described method of creating a vector PT14SS1SG.

E. coli BL21(DE3) (Stratagene, USA) transform vector pT14SS1SG and get the transformant, designated as E. coli HM 10310.

(Stage 4) create a vector containing a gene that encodes a protein STII/hG-CSF

First, the codon-modified gene hG-CSF plasmids pT14SS1SSG, obtained in stage 3, is replaced by Thr by site-directed mutagenesis (Papworth, S. et al., Strategies, 9, 3 (1996)), carrying out PCR in respect of plasmids using the new semantic priming (SEQ ID NO: 13) and pfu (Stratagene, USA).

Allocate amplificatory DNA fragment and add to it the restriction enzyme Dpnl to remove the untransformed plasmids.

E. coli XL-1 blue (Novagen, USA) transforming the modified plasmid. Produce a sequence of bases of DNA isolated from transformed colonies, and thus receive the plasmid pT14SSG, which contains a gene having a Thr instead of the first amino acid of hG-CSF (SEQ ID NO: 11).

E. coli BL21(DS3) (Stratagene, USA) transform vector pT14SSG and get the transformant, designated as E. coli HM 10301.

(Stage 5) create a vector containing the gene encoding the modified STII/hG-CSF

Vector pT14SSG obtained in stage 4, is subjected to PCR using complementary priming SEQ ID NOS: 15 and 16, which are intended to replace the 4th codon STII a Thr codon in accordance with the method described in stage 4, and receive a modified plasmid.

E. coli XL-1 blue (Novagen, USA) transforming the modified plasmid. Produce a sequence of bases of DNA isolated from transformed colonies, and thus receive the plasmid that contains a gene having a Thr instead of the 4th amino acid STII (SEQ ID NO: 14).

(Stage 6) create a vector containing the gene encoding the modified STII/hG-CSF

Vector pT14SSG-4T obtained at stage 5, is subjected to PCR using complementary priming SEQ ID NOS: 18 and 19, which are designed to replace the 22-th codon STII a Gln codon in accordance with the method described in stage 4, and receive a modified plasmid.

E. coli XL-1 blue (Novagen, USA) transforming the modified plasmid. Produce a sequence of bases of DNA isolated from transformed colonies, and thus receive the plasmid pT14SSG-4T22Q, which contains a gene that has a Gln instead of the 22nd amino acids STII (SEQ ID NO: 17).

(Stage 7) create a vector containing the modified sequence STII SD and the gene encoding the modified STII/hG-CSF

Vector pT14SSG-4T22Q obtained at stage 6, is subjected to PCR using complementary priming SEQ ID NOS: 20 and 21, in accordance with the method described in stage 4, and get a vector 14OSSG-4T22Q with six nucleotide sequences between sequence STII SD (GAGG) and initiating codon STII (modified sequence STII SD SEQ ID NO: 71).

In Fig.5 shows anchorman, denoted as E. coli HM 10302.

Example 3. Create a vector containing the gene encoding the modified hG-CSF

To obtain gene modified hG-CSF, oligomer S1 (SEQ ID NO: 22), having a preferred E. coli codons and Ser instead of the 17-th amino acid of hG-CSF, and oligomer AS1 (SEQ ID NO:) is synthesized in a DNA synthesizer (model 380V, Applied Biosystem, USA).

The oligonucleotides in the amount of 0.5 μl (50 pmol) is subjected to interaction with 95C for 15 minutes and incubated for 3 hours until a temperature of 35C. the Mixture was precipitated in ethanol and subjected to gel electrophoresis (SDS-PAGE), while receiving double-stranded oligomer with "sticky" ends.

Plasmid pT14SS1SG, obtained in stage 3 of example 2, disintegrated with ApaI and BstXI and then are ligated with a double-stranded oligomer with "sticky" ends, receiving vector PT14SS1S17SEG. Vector pT14SS1S17SEG contains the gene encoding hG-CSF, which has a preferred E. coli codons of aminocore and Ser instead of 1-th and 17-th amino acid of hG-CSF.

In Fig.6 shows the above-described method of creating a vector pT14OSS1S17SEG.

E. coli BL21(DE3) transformed with vector pT14SS1S173EG and get the transformant, designated as E. coli HM 10311, which were deposited in the Korean Central registry is rasego gene encoding the signal peptide OmpA of E. coli and modified hG-CSF

A vector containing the gene encoding the promoter TAS and signal peptide OmpA (SEQ ID NO: 24), and the gene encoding the modified hG-CSF, was obtained as follows:

The restriction site HindIII

The vector pT-CSF obtained in example 1 is subjected to PCR using the seed (SEQ ID NO: 27), designed to replace the 1st codon hG-CSF by the Ser codon, and the other seed (SEQ ID NO: 28), designed to produce a restriction site EcoRI (5'-GAATTC-3') below the termination codon, and get a DNA fragment containing the gene encoding the modified hG-CSF.

DNA fragment digested using HindIII and EcoRI and then injected at the site HindIII/EcoRI vector pFlag.CTS (Eastman, USA), obtaining the vector pT1SG, which contains the gene encoding the signal peptide OmpA of E. coli and modified hG-CSF (SEQ ID NO: 29).

In Fig.7 shows the above-described method of creating a vector pTO1SG.

E. coli BL21(DE3) (Stratagene, USA) transform vector pTOlSG and get the transformant, designated as E. coli NM 10409.

Example 5. Create a vector containing the gene encoding the signal peptide OmpA of E. coli and modified hG-CSF

The first codon of the gene of the modified hG-CSF plasmids pTO1SG obtained in example 4, replacing Thr put the example 4, using semantic priming (SEQ ID NO: 30), designed to replace the 1st codon hG-CSF by Thr codon and complementary antisense seed (SEQ ID NO: 31).

E. coli XL-1 blue (Novagen, USA) transforming the modified plasmid. Produce a sequence of bases of DNA isolated from transformed colonies, and thus receive the plasmid pTG, which contains a gene having a Thr instead of the first amino acid of hG-CSF.

E. coli BL21(DE3) (Stratagene, USA) transform vector pTOG and get the transformant, designated as E. coli NM 10401.

Example 6. Receiving the modified hG-CSF

(a) Obtaining [Ser1, Serl7] hG-CSF

Vector pTO1SG obtained in example 4 is subjected to PCR using semantic priming (SEQ ID NO: 32), intended to replace the 17-th codon hG-CSF by the Ser codon, and a complementary antisense seed (SEQ ID NO: 33), in accordance with the method described in stage 4 of example 2, and receive a modified plasmid.

E. coli XL-1 blue (Novagen, USA) transforming the modified plasmid. Produce a sequence of bases of DNA isolated from transformed colonies, thus obtaining plasmid pTO1S17SG, which contains a gene having a Ser instead of 1-th and 17-th amino acid of hG-CSF.

E. coli BL21(DE3) (Strata the IAOD in the Korean Central register of cultures of microorganisms (xsm) on March 24, 1999 under access number xsm-10151.

(b) Obtaining [Ser17] hG-CSF

Vector pTOG obtained in example 5 is subjected to PCR using semantic priming (SEQ ID NO: 32), intended to replace the 17-th codon hG-CSF by the Ser codon, and a complementary antisense seed (SEQ ID NO: 33), in accordance with the method described in stage 4 of example 2, and receive a modified plasmid.

E. coli XL-1 blue (Novagen, USA) transforming the modified plasmid. Produce a sequence of bases of DNA isolated from transformed colonies, thus obtaining plasmid pTO17SG, which contains a gene having a Ser instead of the 17-th amino acid of hG-CSF.

E. coli BL21(DE3) (Stratagene, USA) transform vector pTO17SG and get the transformant, designated E. coli HM 10411, which were deposited in the Korean Central register of cultures of microorganisms (xsm) March 24, 1999, under access number xsm-10152.

(C) Obtaining [Thrl7] hG-CSF

Vector pTOG obtained in example 5 is subjected to PCR using semantic priming (SEQ ID NO: 34), intended to replace the 17-th codon hG-CSF by Thr codon and complementary antisense seed (SEQ ID NO: 35), in accordance with the method described in stage 4 of example 2, and receive a modified plasmid.

E. coli XL-1 blue (Novagen, Zelenoy of transformed colonies, thus obtaining plasmid pTO17TG, which contains a gene having a Thr instead of the 17-th amino acid of hG-CSF.

E. coli BL21(DE3) (Stratagen, USA) transform vector pTO17TG and get the transformant, referred to as E. coli HM 10413.

(d) Obtaining [AA] hG-CSF

Vector pTOG obtained in example 5 is subjected to PCR using semantic priming (SEQ ID NO: 36), intended to replace the 17-th codon hG-CSF by codon A1A, and a complementary antisense seed (SEQ ID NO: 37), in accordance with the method described in stage 4 of example 2, and receive a modified plasmid.

E. coli XL-1 blue (Novagen, USA) transforming the modified plasmid. Produce a sequence of bases of DNA isolated from transformed colonies, thus obtaining plasmid pT17AG, which contains a gene having l instead of the 17-th amino acid of hG-CSF.

E. coli BL21(DE3) (Stratagene, USA) transform vector pT17AG and get the transformant, referred to as E. coli HM 10414.

(e) Obtaining [Gly17] hG-CSF

Vector pTOG obtained in example 5 is subjected to PCR using semantic priming (SEQ ID NO: 38), intended to replace the 17-th codon hG-CSF by a Gly codon and complementary antisense seed (SEQ ID NO: 39), in accordance with the method described in stage 4 of example 2, and get modi is the sequence of DNA bases, isolated from transformed colonies, thus obtaining plasmid pTO17GG, which contains a gene with Gly instead of the 17-th amino acid of hG-CSF.

E. coli BL21 (DE3) (Stratagene, USA) transform vector pTO17GG and get the transformant, referred to as E. coli NM 10415.

(f) Obtaining [Asp17] hG-CSF

Vector pTOG obtained in example 5 is subjected to PCR using semantic priming (SEQ ID NO: 40), designed to replace the 17-th codon hG-CSF by the Asp codon and complementary antisense seed (SEQ ID NO: 41), in accordance with the method described in stage 4 of example 2, and receive a modified plasmid.

E. coli XL-1 blue (Novagen, USA) transforming the modified plasmid. Produce a sequence of bases of DNA isolated from transformed colonies, thus obtaining plasmid pT17APG, which contains a gene with Asp instead of the 17-th amino acid of hG-CSF.

E. coli BL21(DE3) (Stratagene, USA) transform vector pT17APG and get the transformant, referred to as E. coli NM 10416.

Example 7. Create a vector containing the gene encoding the signal peptide Gene III of E. coli and modified hG-CSF

(a) create a vector containing the gene encoding arabinosyl the promoter and signal peptide Gene III of E. Coli.

A vector containing the gene encoding eraut as follows:

The restriction site NcoI

Plasmid pBAD gIIIA (Invitrogen, USA) containing the gene encoding arabinosyl the promoter and signal peptide Gene III digested using NcoI, single-stranded DNA can be removed by using DNA polymerase maple and get double-stranded DNA with "blunt" ends, which then disintegrated with BgIII, while receiving a fragment of the vector with the "blunt" end and a "sticky" end.

The vector pT-CSF obtained in example 1 is subjected to PCR using semantic priming (SEQ ID NO: 46), having a nucleotide sequence encoding a 2nd to 9th amino acids of hG-CSF (SEQ ID NO: 45), and a complementary antisense seed (SEQ ID NO: 47), in accordance with the method described in stage 4 of example 2, and get a DNA fragment with the "blunt" ends, containing the gene hG-CSF and the restriction site BamHI at the carboxyl end. The resulting fragment is then digested using BamHI, receiving a fragment of the gene hG-CSF with the "blunt" end and a "sticky" end.

A fragment of the gene hG-CSF is inserted into the above vector, thus obtaining a vector pBADG, which contains the gene encoding the signal peptide Gene III of E. coli and hG-CSF (SEQ ID NO: 48).

In Fig.8 shows the above-described method of creating a vector pBADG.

E. coli BL is giving [Met2, Val3] hG-CSF

Plasmid pBAD gIIIA (Invitrogen, USA) digested using NcoI and BgIII and get the fragment that has two "sticky" ends.

The vector pT-CSF obtained in example 1 is subjected to PCR using semantic priming (SEQ ID NO: 50), having a nucleotide sequence encoding a 1st to 9th amino acids [Met2, Val3] hG-CSF (SEQ ID NO: 49), and a complementary antisense seed (SEQ ID NO: 51), in accordance with the method described in stage 4 of example 2, and get a DNA fragment with the "blunt" ends, containing the gene hG-CSF and the restriction site BamHI at the carboxyl end, which then disintegrated with NeoI and BamHI, thus obtaining a fragment of the gene hG-CSF having two "sticky" ends.

The restriction site NcoI

A fragment of the gene hG-CSF is inserted into the above vector and the receive vector pBAD2M2VG containing the gene encoding the signal peptide Gene III of E. coli, and Met and Val instead of 2nd and 3rd amino acid of hG-CSF (SEQ ID NO: 52).

In Fig.9 shows the above-described method of creating a vector pBAD2M3VG.

E. coli BL21(DE3) (Stratagene, USA) transform vector pBAD2M3VG and get the transformant, designated as E. coli HM 10510, which were deposited in the Korean Central register of cultures of microorganisms (xsm) March 24, 1999, under access number xsm-10153.

(C) Receipt is designated to replace the 17-th codon hG-CSF by codon Ser, and complementary antisense seed (SEQ ID NO: 33), in accordance with the method described in stage 4 of example 2, and receive a modified plasmid.

E. coli XL-1 blue (Novagen, USA) transforming the modified plasmid. Produce a sequence of bases of DNA isolated from transformed colonies, thus obtaining plasmid pBAD17SG, which contains a gene having a Ser instead of the 17-th amino acid of hG-CSF.

E. coli BL21(DE3) (Stratagene, USA) transform vector pBAD17SG and get the transformant, designated as E. coli HM 10511.

(d) Obtaining [Met2, Val3, Serl7] hG-CSF

Vector pBAD2M3VG obtained in paragraph (b), is subjected to PCR using semantic priming (SEQ ID NO: 32), intended to replace the 17-th codon hG-CSF by the Ser codon, and a complementary antisense seed (SEQ ID NO: 33), in accordance with the method described in stage 4 of example 2, and receive a modified plasmid.

E. coli XL-1 blue (Novagen, USA) transforming the modified plasmid. Produce a sequence of bases of DNA isolated from transformed colonies, thus obtaining plasmid pBAD2M3V17SG, which contains a gene with Met, Val, and Ser, respectively, instead of the 2nd, 3rd and 17th amino acids of hG-CSF.

E. coli BL21(DE3) (Stratagene, USA) Transfo sormanti, obtained in examples 2-7, cultivated in LB medium (1% bacteriophora, 0,5% backdragging extract and 1% NaCl) and then incubated in the presence of inducer of the expression (IPTG) for 3 hours or grown without IPTG for more than 15 hours. All cultures are centrifuged at 6000 rpm for 20 minutes to precipitate bacterial cells, after which the precipitate is suspended in 1/10 volume of isotonic solution (20% sucrose, 10 mm Tris-CL buffer solution containing 1 mm EDTA, pH 7.0). The suspension is left to stand at room temperature for 30 minutes, then centrifuged with a speed of 7000 rpm for 10 minutes and collect the bacterial cells. The obtained cells are again suspended in distilled water at 4C, centrifuged with a speed of 7000 rpm for 10 minutes and supernatant get in the form of a solution of periplasm. The content of hG-CSF in solution periplasm determined by ELISA method (Kato, K. et al., J. Immunol., 116, 1554 (1976)), using antibody against hG-CSF (Aland, USA), which is calculated as the number hG-CSF produced in a 1 l culture. The results are shown in the table.

Example 9. Purification of hG-CSF

The transformant E. coli NM 10411 obtained in Priyut and collect cells. Performing the method according to example 8, from cells receive the solution periplasm.

The solution periplasm adjusted to pH 5.0-5.5, adsorb in a column of CM-separate (Pharmacia Inc., Sweden), previously equilibrated to a pH of 5.3, and wash the column with 25 mm NaCl. hG-CSF elute sequentially adding the column buffer solutions containing 50 mm, 100 mm and 200 mm NaCl, and fractions containing hG-CSF, gather and unite.

Combined fractions is subjected to chromatography on columns with phenyltetrazol (Pharmacia Inc., Sweden), while receiving [Ser17] hG-CSF with a purity of 99%.

Further, the above method is performed using the transformed E. coli NM 10311, NM 10409, NM 10411, NM 10413, NM 10414, NM 10415, NM 10510 and NM 10512, obtained respectively in examples 3, 4, 6(b), 6(C) 6(d) 6(e), 7(b) and 7(d).

Each purified fraction of hG-CSF is subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) to determine purity and approximate concentrations of hG-CSF and then analyzed using ELISA method to determine the exact concentration of hG-CSF in solution periplasm. As a control substance use Met-hG-CSF (Kirin amgen).

In Fig.10A shows the results of SDS-PAGE, where lane 1 corresponds Met-G-CSF, lane 2 corresponds to the solution of transformant E. coli NM 10411 in periplasm and dalarnas mass of hG-CSF wild type and solution transformant E. coli NM 10411 in periplasm characterized by high content [Ser17] hG-CSF.

In addition, make the determination of N-terminal amino acid sequences of different hG-CSF and the nucleotide sequences encoding the 1st to 32nd amino acids, obtained from transformants NM 10311, NM 10409, NM 10411, NM 10413, NM 10414, NM 10415, NM 10510 and NM 10512 shown respectively in SEQ ID NOS: 56, 58, 60, 62, 64, 66, 68 and 70. The result shows that the modified hG-CSF of the present invention does not have a methionine residue at the N-Terminus.

Nitrocellulose filter (Bio-Rad Lab., USA) moisten buffer for blotting (170 mm glycine, 25 mm Tris HCl (pH 8), 20% methanol) and the proteins separated on the gel, and analyzed by the method of Western blotting on a nitrocellulose filter (Bio-Rad Lab., USA) for 3 hours. The filter is incubated in 1% casein for 1 hour, and three times washed with PBS containing 0.05% Tween 20. The filter is placed in a solution of goat anti-G-CSF antibody (R&D System, AB-214-NA, USA), diluted with PBS, and subjected to interaction at room temperature for 2 hours. After the reaction, the filter is washed three times with PBST solution to remove unreacted antibody. Add conjugated with horseradish peroxidase rabbit antibodiesa I washed with PBST and add a solution of peroxidase from a set of substances (Bio-Rad Lab., USA) for the implementation of staining. Results above the Western blot shown in Fig.10b, where lane 1 corresponds to the positive control, Met-G-CSF, and track 2 is cleared [Ser17] hG-CSF. As shown in Fig.10b, molecular weight [Ser17] hG-CSF is equal to the molecular mass of hG-CSF wild type.

Example 10. Cellular activity of hG-CSF and the modified hG-CSF

Cell line HL-60 (ATCC CCL-240, obtained from bone marrow white 36-year-old woman, suffering promyelocytic leukemia) were cultured in medium RPMI 1640 containing 10% fetal calf serum, adjusted to 2.2105cells/ml and add DMSO (dimethyl sulfoxide, with a degree of purity for cultivation/SIGMA) to a concentration of 1.25% (vol./vol.). In a 96-well plate (Corning/96-well plate with a low degree of evaporation) add 90 ál of the resulting solution at 2104cells/well and incubated at 37C and 5% CO2within 48 hours.

All modified [Ala17] hG-CSF, [Gly17] hG-CSF, [Ser17] hG-CSF and [Thr17] hG-CSF was diluted in RPMI media 1640 to a concentration of 500 ng/ml and 10-fold serially diluted twofold amount of medium RPMI 1640.

The resulting solution is added to the wells in an amount of 10 the use of commercially available hG-CSF (Jeil Pharmaceutical).

Increased concentration cell line determined using commercially available titer cells CellTiter96(catalog No. G4100, Promega), on the basis of the measured optical density at 670 nm.

As shown in Fig.11, the cellular activity of the modified hG-CSF of similar or higher than the positive control substance, hG-CSF wild type.

It is obvious that in addition to the described and illustrated embodiments of this invention various changes and modifications within the present invention, which is limited only by the scope of the attached claims.

Budapest Treaty on the international recognition of the registration of microorganisms during the patent examination given at the end of the description.

The list of sequences is given at the end of the description.

Claims

1. Modified colony-stimulating factor granulocyte person (hG-CSF), amino acid sequence which corresponds to that of wild-type (SEQ ID NO:2), except that (a) 1-I amino acid is Ser; (b) 1-I amino acid is Ser and 17th amino acid is Ser; (C) 2-I amino acid is Met and 3rd aminati (f) 17-I amino acid is Thr.

2. DNA encoding the modified hG-CSF having a nucleotide sequence that determines the amino acid sequence of the modified hG-CSF under item 1.

3. DNA under item 2, characterized in that the nucleotide sequence 1-60 DNA modified hG-CSF corresponds to the nucleotide sequence selected from the group consisting of SEQ ID NO:55, 57, 61, 67 and 69.

4. Expressing the vector for E. coli containing DNA under item 2, and polynucleotide that encodes a signal peptide selected from the group consisting of a signal peptide thermoresistances enterotoxin II E. coli, modified signal peptide thermoresistances enterotoxin II E. coli, the signal peptide of beta-lactamase E. coli, modified signal peptide beta-lactamase E. coli signal peptide Gene III E. coli or modified signal peptide Gene III E. coli attached at the 5’-end of the DNA encoding the modified hG-CSF.

5. Expressing the vector in p. 4, characterized in that the signal peptide thermoresistances enterotoxin II E. coli has the amino acid sequence of SEQ ID NO:53.

6. Expressing the vector in p. 4, characterized in that the modified signal peptide thermoresistances enterotoxin II is then additionally includes a modified Shine-dalgarno sequence of enterotoxin II E. coli, having the nucleotide sequence of SEQ ID NO:71.

8. Expressing the vector in p. 4, characterized in that the signal peptide of beta-lactamase E. coli has the amino acid sequence of SEQ ID NO:24.

9. Expressing the vector in p. 4, characterized in that the signal peptide Gene III E. coli has the amino acid sequence of SEQ ID NO:42.

10. Expressing the vector in p. 4, characterized in that it is a pT14SS1SG, pT14SS1S17SEG, pT1SG, pT1S17SG, pT17SG or pBAD2M3V17SG.

11. Expressing the vector in p. 4, characterized in that it is used for injection into the organism.

12. Expressing the vector on p. 11, characterized in that the microorganism is a transformed E. coli.

13. Expressing the vector under item 12, characterized in that the transformed E. coli is an E. coli BL21(DE3)/pT14SS1SG(HM 10310), E. coli BL21 (DE3)/pT14SS1S17SEG(HM 10311, xsm-10154), E. coli BL21(DE3)/pT1SG (HM 10409), E. coli BL21 (DE3)/pT1S17SG (HM 10410, xsm-10151), E. coli BL21(DE3)/pT17SG (HM 10411, xsm-10152), E. coli BL21 (DE3)/pTO17TG (HM 10413), E. coli BL21 (DE3)/pT17AG (NM 10414), E. coli BL21 (DE3)/pT17GG (NM 10415), E. coli BL21 (DE3)/pBAD2M3VG (NM 10510, xsm-10153), E. coli BL21(DE3)/pBAD17SG (NM 10511) or E. coli BL21 (DE3)/pBAD2M3V17SG (NM 10512).

14. The method of obtaining modified hG-CSF in a microorganism comprising culturing a microorganism, transform the

 

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