Leu-hirudine precursor and method for preparing leu-hirudine

FIELD: biotechnology, microbiology, medicine.

SUBSTANCE: method involves selection of signal sequence suitable for the effective expression of Leu-hirudine in E. coli cells by the polymerase chain reaction-screening method. Method involves construction of a protein as a precursor of hirudine based on the selected signal sequence of surface membrane protein from Serratia marcescens, oprF protein from Pseudomonas fluorescens or fumarate reductase from Shewanella putrifaciens by joining the Leu-hirudine amino acid sequence with C-end of selected signal sequence. Prepared precursor of Leu-hirudine is used in a method for preparing Leu-hirudine. Invention provides preparing Leu-hirudine by the direct secretion in E. coli cells with the high yield. Invention can be used in preparing the hirudine precursor.

EFFECT: improved preparing method.

4 cl, 1 dwg, 2 tbl, 12 ex

 

Obtained from leeches drug Refludan® shows good therapeutic properties in a clinical trial (The Lancet, Vol.353, p.429-438). This allows to conclude that in the future we should expect needs more of this drug. Biologically active substance of this drug is described in European patent 0324712 [Leu1, Thr2]-63-desulfatohirudin, hereinafter abbreviated as "Leu-hirudin".

In the European patent 0448093 describes a method for hirudin. A preferred variant of the method according to this patent includes hirudin, N-terminal amino acid is alanine. When you merge this with hirudin signal sequence α-cyclodextrines-transferase (CG) and transformation encoding this protein expression vector, as described in this patent, in secretory mutants of E.coli can get Ala-hirudin with the release of the crude product of more than 2 g per liter. In the European patent 0549915 described variants Ala-hirudin with improved stability. Upon receipt of these options using the secretory system of E.coli receive the outputs of several grams per liter. Thus, these outputs are clearly higher than the outputs described Dodt et al. for hirudin variant HV1 (FEBS LETTERS vol.202 373-377, 1986). In U.S. patent 5573929 described messestand the e in comparison with the increase of output at the expression, instead derived from pBR322 vectors Dodt et al., expression cassettes in a known manner through the vector of Fig. Bender et al. (Appl. Environ. Biotechnol. 34, p.203-207, 1990) describe the secretion is described in European patent 0171024 Thr-hirudin using Streptomyces lividans. But also here the outputs are compared with the outputs in the European patent 0448093 and 0549915 are clearly lower. This also applies to the expression in E. coli, which was observed by R. de Taxis du Poet et al. for secretion of hirudin variant HV1 using the signal sequence of the E. coli Ompa. These authors found the outputs of 300 mg/l of hirudin in periplasm and approximately 40 mg/l in the supernatant of cells. At the same time described in this article, the expression in cellular systems insects is low (400 g/l).

Achieved with the use of expression systems, yeast Hansenula polymorpha or Pichia pastoris outputs more just approach described in the European patents 0448093 and 0549915 outputs, as opposed to outputs using S. cerevisiae.

Rosenfeld et al. (Protein Expression and Purification 8, 476-482, 1996) describe the expression and secretion of hirudin yeast Pichia pastoris. Achieved outputs approximately 1.5 g/l culture broth. Similar orders of magnitude can be achieved with the yeast Hansenula polymorpha (Appl. Environ. Biotechnol. 44, 377-385, 1995). However, a significant drawback of such expression systems are clearly the more extended periods of time fermentation in comparison with the system of E.coli. It would also be preferable receipt of Leu-hirudin as Ala-hirudin, through the secretion of using E.coli.

However, this cannot be done is described in the European patent 0448093 system. Therefore, in this patent it is proposed to extend the sequence Leu-hirudin by the Tripeptide Ala-Thr-Arg, and there is a pre-Leu-hirudin, which in the end turn of trypsin in the native active substance Leu-hirudin. If this suggestion is followed, it is found clearly lower outputs of the crude product already in the experiment with shake flasks than outputs described for Ala-hirudin. Thus, a clear advantage in comparison with the later yeast expression systems is not obvious.

Thus, underlying the present invention, the goal was to get protein, in which the combination of signal sequence and Leu-hirudin enables straight-through processing to Leu-hirudin and the subsequent secretion of the native Leu-hirudin in high yields by E.coli. This is a prerequisite for the development of the method, as in fermentation and subsequent purification due to improved initial concentration of hirudin has a positive effect on the cost of obtaining Refludan.

Unexpectedly, it was found that there are signals the s sequence, which make possible direct secretion of Leu-hirudin by E. coli and that there is an even more efficient secretion than is described in the European patent 0448093. As a result, you develop a way that without great expense to produce high quantities of Leu-hirudin. This method is the subject of this invention.

To find the preferred signal sequences using a method based on PCR screening signal sequences. This method uses DNA encoding the target protein, as a matrix and a specific reverse PCR primer, and a variable direct primer, which make possible the synthesis of the DNA fragment that encodes the signal sequence associated with the gene of interest. This reaction proceeds according to the drawing the diagram. Expert it is clear that the number of reaction stages may vary depending on the length of the synthesized signal sequence. A short signal sequence can be obtained with a single stage reaction, a larger sequence with two, three reactions or with a large number of reactions. In addition, the number of reactions depends on the instrument used for the synthesis to be used as the primer of the oligonucleotide. Synthesized so is m follows merged design the sequence of the signal peptide gene can then aim to split recognize cleavage sites 1 and 2 enzymes and built in respectively expressing outdoor vector. This system is a system of shared values in the case, if the target gene is selected hirudin. This N-terminal amino acid of hirudin can be variabelno selected. Although this leads to some effect on the binding of hirudin with thrombin (change the binding constants), but the inhibitory effect of hirudin in relation to the activity of thrombin remains measurable.

In patent application EP-V described the secretion of hirudin in the culture supernatant. There the concentration of hirudin can be directly measured famous test of inhibition of thrombin. The concentration of hirudin is a direct measure for the efficiency of secretion and thereby removal of the signal sequence. However, in this patent is described that, for example, hirudin, beginning with the amino acids leucine, cannot effectively be released in the supernatant by means of the signal sequence CG. Using this process, you can search signal sequences that allow you to do this effectively. This way, you can explore the secretion of hirudinea that begin with one of the other 19 amino acids. So get in each case, the spectrum of the signal sequences, which, in the form of the model allows efficient processing of carboxykinase amino acids si the IGF peptide attached thereto a peptide residue. Thereby, it is possible to perform a preliminary selection of signal peptides for efficient secretion of any protein in periplasm and increase thus the chance of the development of the preferred method of receipt for a particular protein. It is also an object of the present invention. This method can be accelerated or automated in such a way that the mixture for transformation from the reaction mixture for ligation and competent cells in liquid shake culture in selective medium overnight and the next day inoculant an aliquot of these cells environment, for example, described in example 11, which contains a coil for the induction, but most of the culture is centrifuged and the sediment cells frozen. If you detect the expression activity of hirudin, can appropriate expression plasmid re-isolated from these cells, linearize and gel electrophoresis to separate from the possible products autolysosome. Then the linear plasmid DNA are ligated again and again transform into a strain-host. You can select individual colonies and tested for performance expression. This can be achieved in this way will satisfy the eligibility criteria for use as a medicine.

Another advantage of this way of action is the her in different variants of the signal peptide, which arose in the course of evolution through the exchange of amino acids between the individual species can be easily investigated in comparison with each other in respect to their suitability for efficient secretion of hirudin.

This method is also preferable in comparison with the use of computer programs, for example, described by Nielsen et al. (Protein Engineering 10, 1-6, 1997), which can be predicted sites cutting between the signal sequence and interest in protein. However, it appeared that the resulting predictions are not in every case be correct, so that the preferred combination can easily be seen. Moreover, there is no relationship between the prediction of the correct processing and actually achievable output.

The object of this invention is the predecessor of hirudin containing a signal sequence selected from the group consisting of signal sequences of surface membrane protein Serratia marcescens, protein oprF Pseudomonas fluorescens, lamb protein In Escherichia coli (encoded by the genome of the receptor lambda (lamB) and futuretrust Shewanella putrifaciens, preferably selected from the group consisting of a signal sequence, a surface membrane protein of Serratia marcescens and futuretrust Shewanella ptrifaciens, to-the end of which is attached with the sequence Leu-hirudin.

As the subject of this invention is a method for Leu-hirudin, which is used as an intermediate stage, the predecessor of hirudin described above, in which

(a) obtain the expression plasmid containing a DNA sequence encoding the precursor of hirudin;

(b) expression plasmid according to (a) Express in a suitable cell is E. coli;

(c) the predecessor of hirudin secreted from E. coli and simultaneously processed and

(d) Leu-hirudin emit directly from the culture medium.

The subject of this invention is also the use of predecessor hirudin, described above, to obtain Leu-hirudin, preferably in the method, such as the above-described method.

As the subject of this invention is a method of obtaining a suitable signal peptide for the secretory expression of any protein in E. coli, in which

(a) hirudin or hirudin derived with antithrombotic action that have a specific amino acid Asxat N-end, which are connected to the N-end to one of the subjects of the signal peptides, Express in E.coli;

(b) the level of expression is determined by measuring the activity of hirudin in the culture supernatant;

(c) study the (a) and (b) repeat with different signal peptides;

(d) suitable signal peptide is chosen by comparison of expression levels, obtained according to stage (b) activity of hirudin.

The subject of this invention is also the use of hirudin or hirudin derived with antithrombotic action that have a specific amino acid Asxat N-end, for receiving a signal peptide, which allows efficient secretion of a protein precursor, consisting of the signal peptide and any other protein with N-terminal amino acid Asxwhile cleavage of the signal peptide of E. coli, in particular, where Asxis a leucine.

As the subject of this invention is a method of obtaining any protein through the secretory expression in E. coli, in which

(a) suitable signal peptide receive by way of obtaining a suitable signal peptide;

(b) constructing a nucleic acid encoding the precursor protein consisting of a suitable signal peptide in (a) and any protein Express in E. coli; and

(c) any protein isolated from the culture supernatant, in particular, in which N-terminal amino acid of the desired protein is leucine and expression design is a nucleic acid which encodes the signal pepti the sequence encodes a signal peptide, selected from the group consisting of signal peptides surface membrane protein Serratia marcescens, protein oprF Pseudomonas fluorescens, lamb protein In Escherichia coli and futuretrust Shewanella putrifaciens.

For example, should be described in the synthesis signal sequences which make possible the efficient synthesis and secretion of Leu-hirudin. It also describes the synthesis of other signal sequences that do not lead to the goal or lead to a goal with worse outcomes in relation to output. While the examples should explain the idea of the present invention by selecting the signal sequences using Leu-hirudin, but should not limit the invention only to these.

The described methods can be used for cleaning of Refludan; this is described, for example, in example 11.

Example 1

Synthesis of fused gene encoding a protein consisting of Leu-hirudin and the signal sequence of surface membrane protein from Serratia marcescens

As the expression plasmids used are described in European patent 0468539 on the shape 1 vector pJF118, as it respect to its skeleton is identical to the one described in European patent 0448093 vector RSM.

As the matrix is used as mentioned in example 1 of European patent 0448093 plasmid RK, which is a sequence of hirudin, the corresponding Euro is Yeysk patent 0171024.

Protein membrane described Braun, G. and Cole, S.T. (Mol. Gen. Genet. 195, 321-328, 1984).

For the synthesis of the desired DNA fragment was received three oligonucleotide sequence.

Oligonucleotide hirrev has the sequence:

5' TTTTTTTAAG CTTGGGCTGC AGGTC 3' [SEQ ID NO:1]

HindIII

This primer hybridizes with a plot 227-210 P.N. presented in table 1 hirudin gene.

Primer smompaf1 has the sequence:

5'-TGGCACTGGC AGGTTTCGCT ACCGTAGCGC AAGCCcttac gtatactgac tgca-3'[SEQ ID NO:2]

This primer hybridizes with nucleotides 1-19 presented in table 1 sequence of hirudin. Hybridoma part of the sequence of the primer represented by small letters. The rest of the series hybridized with section 229 BP-263 BP published Braun, G. and Cole, S.T. (Mol. Gen. Genet. 195, 321-328, 1984).

Primer smompaf2 has the sequence:

5'-ttttttgaat tcATGAAAAA GACAGCTATC GCATTAGCAG TGGCACTGGC AGGTTTC-3'[SEQ ID NO:3]

From position 13 P.N. this sequence primer hybridizes published Braun and Cole sequence 201 BP-245 BP and, therefore, overlaps with the sequence of the primer smompaf2. Position 1-12 this primer contains a site of recognition for restrictase EcoRI and related six T-nucleotides, that is to allow recognition by this enzyme.

In a standard PCR (for example, 94°S: 10", 50°S: 30", 72°S: 45", 25 cycles) with DNA plasmids RC as a matrix, which is described in table 1 sequence, and primers hirrev and smompaf1 sequence of hirudin extend partial bacterial signal sequence. Then the reaction product is transformed in the second PCR as template with primers hirrev and smompaf1 under the same conditions. As a product of the reaction is a DNA fragment that encodes a protein, which consists of the desired elongated signal sequence sequence of hirudin. At the 5'-end is the site of recognition for restrictase EcoRI and 3'-end of a site of recognition for the enzyme HindIII.

The product of the second PCR turn in the reaction mixture for dual splitting both restrictase and embed in the form of the fragment EcoRI-HindIII open both enzymes DNA vector in T4-DNA-ligase reaction. Competent cells of the strain E. coli Mc1061 or secretory mutant WCM100 transform mixture for ligation and multiply under selection pressure on ampicillinsaturday cups. Then the next morning spend the expression according to example 6 in comparison with the expression of Ala-hirudin with a strain of E.coli WCM100/pCM7053. It turned out that the obtained expression is approximately 1.5 times better than in the comparative experiment.

Por what measures 2

Synthesis of fused protein of Leu-hirudin and the signal sequence of the oprF gene product of Pseudomonas fluorescens

The design is in accordance with the procedure outlined in example 1, scheme, except that instead of primers smompaf1/f2 use two new primers that detect respect to their specificity for the gene of hirudin and sequence recognition by the restriction enzyme EcoRI same properties as the primers smompa, but encode the desired signal sequence oprF gene (De, E. et al.: FEMS Microbial. Lett. 127, 267-272, 1995).

Primer pfuf1 has the sequence:

5' GGTTCTCTTA TTGCCGCTAC TTCTTTCGGC GTTCTGGCAc ttacgtatac tgactgca 3'0[SEQ ID NO:4]

Primer pfuf2 has the sequence:

5' ttttttgaat tcatgAAAAA CACCTTGGGC TTGGCCATTG GTTCTCTTAT TGCCGC 3'[SEQ ID NO:5]

When this primer pfuf1 apply accordingly to example 1, in PCR and primer pfuf2 respectively PCR. The expression is carried out in comparison with the expression of Ala-hirudin with a strain of E. coli WCM100/pCM7053. It turned out that the obtained expression is approximately 1.1 times better than in the comparative experience. After separation by gel electrophoresis in the system PAG-ordinator band secrete hirudin and determine the N-terminal sequence of hirudin. It turned out that the same sequence is fully intact and begins with the amino acid leucine. This result is unexpected, as the program identification of the proposed site recognition signal peptidases predicts extension of hirudin with valine.

Example 3

Synthesis of fused protein of Leu-hirudin and the signal sequence of the lamB gene product from E. coli

The design is in accordance with the procedure outlined in example 1, scheme, except that instead of primers smompaf1/f2 use two new primers that detect respect to their specificity for the gene of hirudin and sequence recognition by the restriction enzyme EcoRI same properties as the primers smompa, but encode the desired signal sequence lamb gene (Clement, J.M. and Hofnung, M.: Cell 21, 507-514, 1981).

Primer lambbf1 has the sequence:

5'GTTGCCGTCG CAGCGGGCGT AATGTCTGCT CAGGCAATGG CTcttacgta tactgactgc a 3'[SEQ ID NO:6]

Primer lambbf2 has the sequence:

5' ttttttgaattcATGATGAT TACTCTGCGC AAACTTCCTC TGGCGGTTGC CGTCGCAGC 3'[SEQ ID NO:7]

When this primer lambb1 apply accordingly to example 1, in PCR and primer lambb2 respectively PCR. The expression is carried out in comparison with the expression of Ala-hirudin strain .coil WCM100/pCM7053. It turned out that the resulting expression is the same as Express the comparative experience. After separation by gel electrophoresis in the system PAG-ordinator band secrete hirudin and determine the N-terminal sequence of hirudin. It turned out that this sequence is fully intact and begins with the amino acid leucine. This result is unexpected, as the program identification of the proposed site recognition signal peptidases does not predict the correct processing of hirudin.

Example 4

Synthesis of fused protein of Leu-hirudin and the signal sequence of the precursor of subunit flavoprotein futuretrust from Shewanella putrifaciens

The design is in accordance with the procedure outlined in example 1, scheme, except that instead of primers smompa f1/f2 are two new primers that detect respect to their specificity for the gene of hirudin and sequence recognition by the restriction enzyme EcoRI same properties as the primers smompa, but encode the desired signal sequence of Shewanella putrifaciens (Pealing S.L. et al.: Biochemistry 31, 12132-12140, 1992). As this publication describes only the sequence of the protein, this amino acid sequence is translated, according to the table of codons in the DNA sequence, so got to

primer spfccf1 the following sequence:

5'CTACCCTGAT GGGTACCGCT GGTCTGATGG GTACCGCTGT TGCTcttacg tatactgact gca 3' [SEQ ID NO : 8]

Prime the RA spfccf2 the following sequence:

5'ttttttgaat tcATGAAAAA AATGAACCTG GCTGTTTGCA TCGCTACCCT GATGGGTACC 3' [SEQ ID NO : 9]

When this primer spfccf1 apply accordingly to example 1, in PCR and primer spfccf2 respectively PCR. The expression is carried out in comparison with the expression of Ala-hirudin with a strain of E.coli WCM100/pCM7053. It turned out that the obtained expression is approximately 1.5 times higher than the expression in the comparative experience. After separation by gel electrophoresis in the system PAG-ordinator band secrete hirudin and determine the N-terminal sequence of hirudin. It turned out that this sequence is fully intact and begins with the amino acid leucine. This result is unexpected, as the program identification of the proposed site recognition signal peptidases predicts processing in carboxyfullerene relative to the cysteine at position 6 of the sequence of hirudin.

Example 5

Synthesis of fused protein of Leu-hirudin and the signal sequence of the precursor β-lactamase from pBR322

The design is in accordance with the procedure outlined in example 1, scheme, except that instead of primers smompaf1/f2 use two new primers that detect respect to their specificity for the gene of hirudin and sequence recognition by the restriction enzyme EcoRI same properties as the primers smompa, but encode desire the th signal sequence of the protein precursor β -lactamases (Sutcliffe J.G.: Cold Spring Harbor Symp. Quant. Biol. 43:77-90 (1978)).

Primer blatf1 has the following sequence:

5' CTGATCCCGT TCTTTGCAGC GTTCTGCCTG CCGGTTTTCG CGcttacgta tactgactgc a 3'[SEQ ID NO:10]

Primer blatf2 has the following sequence:

5' tttttgaat tcATGTCCAT CCAGCACTTC CGCGTCGCCC TGATCCCGTT CTTTGC 3'[SEQ ID NO:11]

When this primer blatf1 apply accordingly to example 1, in PCR and primer blatf2 respectively PCR. The expression is carried out in comparison with the expression of Ala-hirudin with a strain of E. coli WCM100/pCM7053. It turned out that the resulting output expression is only 50%-90% obtained in the comparative experience of entering. After separation by gel electrophoresis in the system PAG-ordinator band secrete hirudin and determine the N-terminal sequence of hirudin. It turned out that this sequence is fully intact and begins with the amino acid leucine. This result was predicted by the program to identify the intended site of recognition of the signal peptide.

Example 6

Synthesis of fused protein of Leu-hirudin and the signal sequence of the precursor of alkaline phosphatase from E. coli

The design is in accordance with the procedure outlined in example 1, scheme, except h is about instead primers smompaf1/f2 use two new primer, that shows respect to their specificity for the gene of hirudin and their sequence recognition by the restriction enzyme EcoRI same properties as the primers smompa, but encode the desired signal sequence of the protein alkaline phosphatase from E. coli (Shuttleworth, H., Taylor, J. and Minton, N. Nucleic Acids Res. 14 (21), 8689 (1986)).

Primer linkphoaf1 has the following sequence:

5' GCTGCCGCTG CTGTTCACCC CGGTTACCAA AGCGcttacg tatactgact gca 3'[SEQ ID NO:12]

Primer linkphoaf2 has the sequence:

5' ttttttgAAT TCATGAAACA GTCGACCATC GCGCTGGCGC TGCTGCCGCT GCTGTTC 3'[SEQ ID NO:13].

Both primers are optimized in accordance with the choice of codons for E. coli, i.e. they are not fully correspond to the natural sequence of the original gene.

When this primer linkphoaf1 apply accordingly to example 1, in PCR and primer linkphoaf2 respectively PCR. The expression is carried out in comparison with the expression of Ala-hirudin with a strain of E.coli WCM100/pCM7053. It turned out that the resulting output expression is only a tiny fraction obtained in the comparative experience of entering. After separation by gel electrophoresis in the system PAG-ordinator band secrete hirudin and determine the N-terminal sequence of hirudin. It turned out that this sequence is a Ki is fully intact and begins with the amino acid leucine. This result was predicted by the program to identify the alleged site of the recognition signal peptidases. But the unexpected is a bad idea.

Example 7

Synthesis of fused protein of Leu-hirudin and the signal sequence of the precursor of alkaline phosphatase from E.fergusonii

The design is in accordance with the procedure outlined in example 1, scheme, except that instead of primers smompaf1/f2 use two new primers that detect respect to their specificity for the gene of hirudin and their sequence recognition by the restriction enzyme EcoRI same properties as the primers smompa, but encode the desired signal sequence of the protein alkaline phosphatase of E. fergusonii (Du Bose, R.F. and Hartl, D.L. Mol. Biol. Evol. 7, 547-577 (1990)).

This signal sequence differs in five positions from the signal sequence of alkaline phosphatase of E. coli.

Primer fergusf1 has the following sequence:

5' GCTGAGCTGC CTGATCACCC CGGTGTCCCA GGCGcttacg tatactgact gca 3'[SEQ ID NO:14]

Primer fergusf2 has the sequence:

5' ttttttgaat tcATGAAACA GAGCGCGATC GCGCTGGCTC TGCTgAGCTG CCTGATC 3'[SEQ ID NO:15]

Both primers are optimized in accordance with the choice of codons for E. coli, i.e. they correspond Tvout not completely natural sequence of the original gene. When this primer fergusf1 apply accordingly to example 1, in PCR and primer fergusf2 respectively PCR. The expression is carried out in comparison with the expression of Ala-hirudin with a strain of E.coli WCM100/pCM7053. It turned out that the resulting output expression is only a tiny fraction obtained in the comparative experience of entering. This output is approximately less than half that of the output, which is observed for the design of the signal peptide of alkaline phosphatase of E. coli and Leu-hirudin.

Example 8

Synthesis of fused protein of Leu-hirudin and the signal sequence of the precursor of the cyclodextrin-glucanotransferase from Paenibacillus macerans

The design is in accordance with the procedure outlined in example 1, scheme, except that instead of primers smompaf1/f2 use two new primers that detect respect to their specificity for the gene of hirudin and their sequence recognition by the restriction enzyme EcoRI same properties as the primers smompa, but encode the desired signal sequence of a gene cyclodextrin-glucanotransferase from Paenibacillus macerans (Nakano, T., Fukuda, M., Monma, M., Kobayashi, S., Kainuma, K. and Yamane, K. J. Bacteriol. 166, 1118-1122 (1986)).

Primer baccdgf1 has the following sequence:

5' CTTTCGCTGA GTATGGCGTT GGGGATTTCA CTGCCCGCAT GGGCActtac gtatactgac tgca 3'[SEQ ID NO:16]

Prime is p baccdgf2 has the following sequence:

5' ttttttgaat tcATGAAATC GCGGTACAAA CGTTTGACCT CCCTGGCGCT TTCGCTGAGT ATGGC 3'[SEQ ID NO:17]

When this primer baccdgf1 apply accordingly to example 1, in PCR and primer baccdgf2 respectively PCR. The expression is carried out in comparison with the expression of Ala-hirudin with a strain of E.coli WCM100/pCM7053. It turned out that the resulting output expression is approximately1/4obtained in the comparative experience of entering. This synthetic hirudin behaves in the test of inhibition of thrombin, as Leu-hirudin. This means that the signal peptide was correctly processionals. It is not as expected from theoretical analysis, which indicated lengthened by 8 amino acids or alternative shortened to two amino acids of the N-end.

Example 9

Synthesis of fused protein of Leu-hirudin and the signal sequence of the protein precursor fibrillin (fotA) E.coli PCFO20

The design is in accordance with the procedure outlined in example 1, scheme, except that instead of primers smompaf1/f2 use two new primers that detect respect to their specificity for the gene of hirudin and their sequence recognition by the restriction enzyme EcoRI same properties as the primers smompa, but encode the desired signal sequence of the protein precursor fibrillin E. coli PFO20 (Viboud, G.I., Jonson, G., Dean-Nystrom, E. and Svennerholm, A.M. Inaect. Immun. 64 (4), 1233-1239 (1996)).

Primer pcf1-ala has the following sequence:

5' TGGTTTCAGC TTTAGTAAGC GGGGTTGCAT TTGCTCTTAC GTATACTGAC TGCAC 3'[SEQ ID NO:19]

Primer p-pcf2 has the sequence:

5' TTTTGGGAAT TCATGAAAAA GACAATTATG TCTCTGGCTG TGGTTTCAGC TTTAGTAAGC 3'[SEQ ID NO:19]

When this primer pcf1-ala is applied according to example 1, in PCR and primer p-pcf2 respectively PCR. The expression is carried out in comparison with the expression of Ala-hirudin with a strain of E. coli WCM100/pCM7053. It turned out that the resulting output expression is approximately 40% obtained in the comparative experience of the exit.

Example 10

Synthesis of fused protein of Leu-hirudin and the signal sequence of surface membrane protein (fimD) S.typhimurium

The design is in accordance with the procedure outlined in example 1, scheme, except that instead of primers smompaf1/f2 use two new primers that detect respect to their specificity for the gene of hirudin and their sequence recognition by the restriction enzyme EcoRI same properties as the primers smompa, but encode the desired signal sequence of surface membrane protein S.typhimurium (Rioux, C.R., Friedrich, M.J. and Kadner, R.J.: J. Bacteriol. 172 (11), 6217-6222 (1990)).

Primer styfimf1 has the following sequence:

5' CGGCGCTGAG TCTCGCCTTA TTTTCTCACC TATCTTTTGC Ccttacgtat actgactgca 3'[SEQ ID NO:20]

Primer styfimf2 has the sequence:

5' ttttttgaat tcaTGTCATT TCATCACCGG GTATTTAAAC TGTCGGCGCT GAGTCTC 3'[SEQ ID NO:21]

When this primer styfimf1 apply accordingly to example 1, in PCR and primer styfim2 respectively PCR. The expression is carried out in comparison with the expression of Ala-hirudin with a strain of E. coli WCM100/pCM7053. It turned out that the resulting output expression is approximately 10% obtained in the comparative experience of the exit.

Example 11

Expression in E. coli

This example describes the expression of hirudin. For this purpose, in each case 1-5 ml LB-medium, which contains 25 mg/l of ampicillin and 0.5-2 mm IPTG (isopropyl-β-D-thiogalactopyranoside) inoculant cells transformant and shaken for approximately 20 hours at 28°in an incubation shaker at 220 rpm Then after determining the optical density of the cell suspension centrifuged and determine hirudin from the clear supernatant.

In parallel, expression of refludan spend the expression described in the European patent 0448093 Ala-hirudin by plasmids RSM described in this patent secretory mu the ante WCM100. This allows a direct comparison of the expression level.

The expression on a larger scale can be carried out in accordance with U.S. patent 5616476. Refludan can then be purified as described in examples 5 and 6 in this patent ways.

Example 12

Determining the concentration of hirudin

Determining the concentration of hirudin is carried out in accordance with the method Griessbach et al. (Thrombosis Research 37, 347-350, 1985). For this purpose, a certain number of standards of Refludan include to obtain the calibration curve in the range measurements. Due to this, the output is directly in mg/L.

1. The predecessor of hirudin containing a signal sequence selected from the group consisting of signal sequences of surface membrane protein Serratia marcescens, protein oprF Pseudomonas fluorescens and futuretrust Shewanella putrifaciens, With the end which is attached with the sequence Leu-g is Rudin.

2. The predecessor of hirudin according to claim 1, in which the signal sequence is selected from the group consisting of a signal sequence, a surface membrane protein of Serratia marcescens and futuretrust Shewanella putrifaciens.

3. The predecessor of hirudin on one of paragraphs 1 or 2 used to produce Leu-hirudin.

4. The method of obtaining Leu-hirudin, namely, that create the expression plasmid containing a DNA sequence encoding the precursor of hirudin according to claim 1, expression plasmid Express in a suitable cell is E. coli and Leu-hirudin emit directly from the culture medium.



 

Same patents:

FIELD: gene and protein engineering for various luminescent assays.

SUBSTANCE: new luciferase mutant forms have been obtained. Said mutant forms have increased thermostability and optionally different emission wave length in contrast with respective wild type enzymes. In all disclosed muteins natural amino acid residue in position equivalent to 357-position in Photinus pyralis luciferase sequence is replaced with other residue, preferably with uncharged polar amino acid (in particular tyrosine) residue. Mutant luciferases of present invention are useful in various analytical systems as reporter agent.

EFFECT: Mutant luciferases with new properties.

22 cl, 15 dwg, 6 tbl, 12 ex

FIELD: genetic engineering, biotechnology, biochemistry, agriculture, food industry, medicine.

SUBSTANCE: invention relates to the transformation of plant with nucleic acid encoding enzyme Δ6-desaturase in C. elegans that results to preparing a plant with enhanced content of gamma-linolenic acid and resistance to cold. Desaturase extracted from the plant can be used for preparing a drug used for treatment of disorder in body associated with deficiency of gamma-linolenic acid in it.

EFFECT: valuable biological properties of genes and desaturases.

36 cl, 9 dwg, 2 ex

The invention relates to biotechnology and Cryobiology

The invention relates to genetic engineering and can be used in plant breeding

The invention relates to biotechnology

The invention relates to biotechnology and can be used in the baking industry

The invention relates to biotechnology and can be used in the baking industry

The invention relates to medicine, more specifically to methods of treatment using medicinal electrophoresis, namely for the treatment of acne and diseases of the spine, in particular to methods for treating a compression-ischemic plexopathy

FIELD: biotechnology, medicine.

SUBSTANCE: Zalpha 11-ligand is isolated from cDNA library generated from activated cells of human peripheral blood that have been selected from CD3. Animal is inoculated with Zalpha 11-ligand and antibodies are prepared that are able to bind specifically with epitopes, peptides or polypeptides of Zalpha 11-ligand. Invention provides effective regulation and/or development of hemopoietic cells in vitro and in vivo. Invention can be used for preparing Zalpha 11-ligand and antibodies for it.

EFFECT: valuable properties of new cytokine.

18 cl, 5 tbl, 1 dwg, 55 ex

FIELD: genetic and tissue engineering, biotechnology, medicine, agriculture.

SUBSTANCE: invention relates to the development of simple with constructive relation peptide vector (PGE-κ) consisting of polypeptide sequence of epidermal growth factor (EGF) and modified sequence of signal peptide T-antigen SV-40. New vector PGE-κ is able to provide the selective delivery of genetic material in target-cell cytoplasm carrying external receptors to EGF and the following its transport across nuclear membrane. Also, invention proposes a method for preparing peptide vector PGE-κ involving its expression as a fused protein "mutant thioredoxine-linker-vector" and cleavage of product expressed in E. coli in the linker region with specific protease. Invention provides preparing the recombinant strain E. coli B-8389 VKPM as a producer of the fused protein comprising PGE-κ. Proposed vector shows simple structure, absence of toxicity and immunogenicity and these properties provide its usefulness for the directed genetic modification of epithelial, embryonic and tumor cells in vivo.

EFFECT: improved preparing method, valuable medicinal properties of vector, improved genetic modification.

7 cl, 12 dwg, 4 tbl, 16 ex

FIELD: genetic engineering, molecular biology.

SUBSTANCE: invention proposes a method for detecting genes encoding membrane-bound transmembrane proteins. Method involves expression of the nucleic acid chimeric sequence in the cell-host consisting of DNA fragment encoding secreting protein that is able to bind antigen and DNA fragment to be tested; interaction of cells expressing the fused protein with antigen; selection of cells on surface of that indicated antigen is bound; isolation of recombinant vector containing in selected cells of DNA fragment to be tested and, if necessary, determination of its sequence. Also, invention proposes the developed vector constructions and comprising their sets designated for realization of the proposed method. Invention provides significant simplifying the screening process of libraries and cloning genes encoding transmembrane proteins. Invention can be used for detecting and preparing genes encoding any membrane-bound proteins used in different branches of science and practice.

EFFECT: improved isolating method, valuable biological properties of protein.

27 cl, 7 dwg, 1 tbl, 8 ex

FIELD: biotechnology, molecular biology, medicine, genetic engineering, pharmacy.

SUBSTANCE: the hemopoietic protein comprises the amino acid sequence of the formula: R1-L1-R1, R2-L1-R1, R1-R2 or R2-R1 wherein R1 represents the modified ligand flt-3; R2 represents the modified human IL-3, the modified or unmodified colony-stimulating factor. Modification of R1 is carried out by addition of N-end with C-end directly or through linker (L2) that is able to join N-end with C-end to form new C- and N-ends. The modified human IL-3 is prepared by replacing amino acids at positions 17-123. The human G-CSF is modified by exchange of amino acids. The hemopoietic protein is prepared by culturing cells transformed with vector comprising DNA that encodes the hemopoietic protein. The hemopoietic protein stimulates producing hemopoietic cells and this protein is used as a component of pharmaceutical composition used in treatment of humans suffering with tumor, infectious or autoimmune disease. Invention provides preparing multifunctional hemopoietic proteins eliciting the enhanced activity with respect to stimulation of hemopoietic cells and eliciting the improved physical indices. Invention can be used for preparing chimeric multifunctional hemopoietic proteins.

EFFECT: improved preparing and producing method, valuable medicinal properties of protein.

22 cl, 19 dwg, 18 tbl, 117 ex

FIELD: genetic engineering, immunology, medicine.

SUBSTANCE: invention relates to new antibodies directed against antigenic complex CD3 and can be used in therapeutic aims. Antibody IgG elicits the affinity binding with respect to antigenic complex CD3 wherein heavy chain comprises skeleton of the human variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 2, 4 and 6 and their corresponding conservatively modified variants. Light chain comprises skeleton of the rodent variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 8, 10 and 12 and their corresponding conservatively modified variants. Antibody is prepared by culturing procaryotic or eucaryotic cell co-transformed with vector comprising recombinant nucleic acid that encodes antibody light chain and vector comprising recombinant nucleic acid that encodes antibody heavy chain. Antibody is administrated in the patient suffering with malignant tumor or needing in immunosuppression in the effective dose. Invention provides preparing chimeric antibodies against CD3 that are produced by expression systems of procaryotic and eucaryotic cells with the enhanced yield.

EFFECT: improved preparing methods, valuable medicinal properties of antibody.

33 cl, 5 dwg, 1 ex

The invention relates to genetic engineering and can be used for therapeutic purposes, in particular in the treatment of neoplastic processes

The invention relates to the field of biotechnology and can be used to obtain a fused protein with increased eritropoetinovmi activity

FIELD: biotechnology, microbiology, vitamins.

SUBSTANCE: method relates to a method for preparing riboflavin by culturing the microorganism Bacillus subtilis as a producer in the nutrient medium containing rib-operon from Bacillus amyloliquefaciens, or microorganism able for utilization of glycerophosphate as a single carbon source, or eliciting the resistance against inhibition of growth by glyoxylate, and extraction of riboflavin prepared. Invention uses the following strains as producers of riboflavin: B. subtilis GM51/pMX45, B. subtilis GM41/pMX45, B. subtilis GM44/pMX45. Invention provides preparing riboflavin of the high degree of effectiveness.

EFFECT: improved preparing method.

5 cl, 4 ex

Up!