Recombinant plasmid dna p ua bc22 encoding a modified plasminogen activator urokinase type, noncoding dna element - artificial intergenic sequence mgp and the bacterial strain escherichia coli producing a modified plasminogen activator urokinase type

 

(57) Abstract:

The invention relates to genetic engineering, in particular to a technology for obtaining high-yielding strains Eserichia coli - produced recombinant human proteins used in modern medicine as thrombolytic agents. The new recombinant plasmid DNA UABC22 encoding a modified plasminogen activator urokinase type (Maputo) has a size of 4.5 T. p. N. and contains: XmnI - BamHI - fragment DNA plasmids U22 size 2,99, etc., ad; BamHI - kpni restriction sites - DNA fragment size 0,177, etc., ad, containing the site of the coding region of the gene Maputo, corresponding to 12 C-terminal amino acids Maputo, and noncoding element - artificial intergenic sequence MGP; Kpn I - SacI - DNA fragment size of 0.57, etc., ad , containing the gene for tRNA AGD; SI - XmnI fragment DNA vector U19 size of 0.79, etc., B.C., site of replication initiation and promotora-operator region l-operon; genetic markers; gene for resistance to ampicillin. Noncoding DNA elements - artificial intergenic sequence MGP and tRNA gene AGD increase the overall stability of the expression system, Maputo in Escherichia coli cells and enhance its broadcasting activity against eukaryotic mainly in preferments form (mol.m. 43 kDa) in the amount of 200000 - 250000 per 1 g of biomass. 3 C. p. F.-ly, 4 Il.

The invention relates to genetic engineering, in particular to a technology for obtaining high-yielding strains of Escherichia coli producing recombinant human proteins used in modern medicine as thrombolytic agents,

It is known that the urokinase extracted from urine, presents three forms: low molecular weight (33 kDa), high-molecular dvuhtsepochnyj (50-55 kDa) and high molecular odnozadachnoy (PUK), with a significant predominance of low molecular weight form. The greatest value for medicine are thrombolytic drugs in the form of high-molecular-weight precursor plasminogen activators, in particular the PUK, due to its low activity until it is activated by a blood clot. The process of obtaining prourokinase from the culture medium of eukaryotic cells-producers is relatively simple, but is disadvantageous because of the high cost of cultivation of cells, which caused the development of genetically engineered approach to solving the problem of production of plasminogen activators urokinase type on the basis of strains of microorganisms.

Known recombinant plasmid pUK54t the IDA (Holmes, W. E., Pennica, D. et al., Cloning and expression of human urokunaze type plasminogen activator in Esherichia coli, Bio Thechnology (1985) V. 3, p. 923-929).

Expression of recombinant urokinase plasmid pUK54trp207-I is carried out with tryptophan promoter after induction intracrinology or endolysosomal acid. The output is about 40% of active urokinase in 1 ml of biomass.

Known recombinant plasmid DNA pUABC encoding a modified plasminogen activator urokinase type (Maputo), and Escherichia coli producing Maputo (RF Patent N 1692151, C 12 N 15/52, 1990).

Plasmid pUABC consists of the following structural elements:

XbaI - BglI fragment (1.39, etc., ad), containing an area of replication initiation and promotora-operator region of the lac-operon vector pUC19;

BglI - > PST fragment (1.28, etc., ad), containing lactamase gene of the vector pUC18;

> PST - XbaI fragment of cDNA of human urokinase (1.44 etc., ad).

Modified plasminogen activator encoded by this plasmid is a recombinant PUK, which differs from the natural prourokinase replacing the first 24 amino acids of the N-terminal domain with homology to epidermal rostovomu factor, 15 alien amino acids.

To construct plasmids pUABC and the (0.42, etc., ad) and EcoRI - > PST (0.60, etc., ad ), the resulting immuno - and oligonucleotide screening of the cDNA library HL1011b of lung fibroblasts on the basis of the vector lambda gt11 (firm CLONTECH, USA). A characteristic feature of this design is a modification of the N-terminal domain of prourokinase, causing a loss of product properties to give the cell mobility, promotora thereby the growth and metastasis of tumors.

Producing strains modified ABOUT VKPM B-4534 obtained by transformation of JM109 Escherichia coli cells with plasmid pUABC. The strain VKPM B-4534 resistant to ampicillin and grown in LB medium gives 2.2 g cell biomass per 1 l of culture medium and produces modified ABOUT mainly in preferments form (mol. m 43 kDa) in the amount of 60,000 units per 1 g of biomass (or 320 units per 1 ml of biomass) in laboratory scale.

However, this producer is not enough stable and productive for large-scale production.

The claimed group of inventions can significantly improve the productivity and stability of the producer strain Maputo.

An increase in the productivity and stability of the producer strain is achieved primarily through changes in noncoding last the coding Maputo, increase overall system stability of expression of Maputo in Escherichia coli cells and enhance its broadcasting activity against eukaryotic genes. Thus, since the modification does not affect coding sequences, all properties of the protein product to be stored, which is confirmed by determining the nucleotide sequence of the gene Maputo and its N - and C-terminal amino acids.

The new recombinant plasmid DNA pUABC22 (Fig. 1) encoding a modified plasminogen activator urokinase type has a size of 4.5 T. p. N. and contains:

- XmnI - BamHI - fragment DNA plasmids pUABC22 size 2.99 T. p. N.;

- BamHI - kpni restriction sites - DNA fragment size 0.177, etc., ad, containing the site of the coding region of the gene Maputo, corresponding to 12 C-terminal amino acids Maputo, and noncoding element - artificial intergenic sequence MGP;

- Cloned - SacI fragment of DNA the size of 0.57, etc., ad, containing the gene for tRNA-Arg.;

- SacI - XmnI fragment DNA vector pUC19 size 0.79 T. p. N.;

- the site of replication initiation and promotora-operator region of the lac operon;

genetic markers;

- gene resistance to ampicillin.

XmnI - BamHI - fragment DNA plasmids pUABC contains 3'-concavo the most part of the coding region of the gene of urokinase, the corresponding sequence Maputo 1 to 389 amino acid.

Gene tRNA Arg encodes the corresponding tRNA and does not encode any protein.

SacI - XmnI fragment DNA vector pUC19 contains a fragment of the gene beta-galactosidase of E. coli and 5'-terminal part of the gene of resistance to ampicillin.

Plasmid pUABC22 obtained by modification of the plasmid pUABC (Fig. 2), namely the introduction of its vector part two noncoding DNA elements: artificial intergenic sequence MGP (0,13, etc., ad), the structure is similar intergenic repeats in the genome of bacteria (IHL sequence) (Cell (1984), V. 37, p. 1015-1026), and the gene sequence of the tRNA-Arg (0,42, etc., ad ), isolated from the chromosome of E. coli by using polymerase chain reaction.

New artificial intergenic sequence MGP shown in Fig.C. This sequence does not encode protein products.

New producing strains of Maputo VKPM B-7666 obtained by transformation of cells of E. coli JM109 two plasmids: pUABC22, coding Maputo, and plasmid pR4, producing additional number of regulatory protein LacI, which also has a stabilization effect. Synthesis of recombinant protein Maputo is under the control of reedy and produces mainly in Maputo preferments form (mol.m. 43 kDa) in the amount of 200 to 250 000 units per 1 g of biomass (3-5 mg per 1 l of culture). Thanks to the use of two noncoding DNA elements, the efficiency of the expression system, Maputo in a new strain-producer of VKPM B-7666 3-4 times higher than in the known strain VKPM B-4534.

To obtain active recombinant Maputo producing strains grown in LB-medium, the cells are precipitated by centrifugation, reveal a treatment with lysozyme and ultrasound, sedimentary fraction of proteins dissolved in guanidinate with 2-mercaptoethanol, diluted and are reconstitution in the presence of denaturing agent guanidinate and oxidized and reduced forms of glutathione, after which the drug cialiswhat. The activity of the product is measured by test fibrinolysis on fibrin plates.

Example 1. Construction of plasmids pUABC22.

Plasmid pUABC hydrolyzing the restriction enzyme AvaII and produce the completion of the sticky ends using fragment maple DNA polymerase I. After phenol deproteinization and resultant deposition rates produce ethanol hydrolysis with restriction enzyme BamHI. After electrophoretic separation of restriction fragments in a 10% polyacrylamide gel fragment length 50 p. N. elute from the gel, purified and CLO is ine restrictive fragment Cloned-SacI. The nucleotide sequence of the insert is determined by the Sanger.

The obtained plasmid pBCOO hydrolyzing restrictase > PST and Cloned and used as a vector sequence. Synthetic oligonucleotides RRE1, RRE2, RRE3 and RRE4, the sequence of which is shown in Fig. 4, phosphorylate with the help of the T4 phage polynucleotide kinase, annealed pairwise and alloyed with the vector using DNA ligase of phage T4. The resulting recombinants identified in length restrictor fragment Cloned-SacI and the nucleotide sequence determined by the Sanger.

The obtained plasmid pRE4 containing a sequence MGP, used to construct plasmids pNO1. This plasmid pUABC hydrolyzing restrictase BamHI and XmnI fragment size 3, etc., N. allocate by electrophoresis in 0.8% agarose gel. Plasmid pUC19 hydrolyzing restrictase Cloned and XmnI fragment size 0.79, etc., N. allocate by electrophoresis in 0.8% agarose gel. Plasmid pRE4 hydrolyzing restrictase and kpni restriction sites BamHI and produce fragment 0.177, etc., called by electrophoresis in 1.2% agarose gel. Obtained three fragments alloyed with the help of DNA ligase of phage T4 and receive plasmid pNO1.

To obtain a fragment of tRNA Arg use p the oligonucleotides YI and Y2:

5'-GCTGATTCAGTCAGGCGTCCCATTATCAGTG-3' and

5'-CAGGAAGGTAAAACCGTCTGTTCCGGGCA-3'

The PCR reaction conditions: concentration of oligonucleotides is 50 uM concentration of each of the 4 deoxynucleotides is 250 uM. Use the buffer HEPES - NaOH (50 mM, pH 8.5), concentration of MgCl2is 1.6 mM. The regime is currently under establishment: 93oC 1 min; then 35 cycles (93oC 30 sec; 67oC 3 min). The PCR product analysis results in a 1% agarose gel has a length of 0.5 T. p. N. the PCR Product by hydrolyzing restrictase SphI and ClaI and produce fragment 0.57, etc., N. using agarose gel electrophoresis. To the obtained fragment attach using DNA ligase of phage T4 flanking sequence oligonucleotides KC1, KC2 and SP1, SP2, respectively

5'-CCGATCCGT-3';

3'-CATGGGCTAGGCAGC-5';

5'-CATGCGGCCGCAATTCGAGCT-3';

3'-GTACGTACGCCGGCGTTAAGC-5'

and clone between areas of recognition of restricted and kpni restriction sites SacI plasmids pNO1. B the result is the plasmid pUABC22. Correct Assembly restricted fragments confirmed by determining the nucleotide sequence according to the method of Sanger.

Example 2. Construction of plasmids pR4.

Plasmid pACYC184 hydrolyzing the restriction enzyme PvuII with the formation of fragments of size 0.5 and 3.7 T. p. N., hydrolyzed alloyed with synthetic Sa is RA fragments of length 0.5, 1.6 and 2.1, etc., ad, and then by the restriction enzyme HindIII with the formation of fragments of lengths 0.5, 1.0, 1.6 and 2.1, etc., called Fragment length 1.0 T. p. N. purified and used in the future. Plasmid pNEO hydrolyzing restrictase SalI and HindIII with the formation of fragments of size 1.8 and 3.7, etc., called Fragment length 1.8 T. p. N. purified and used in the future. The resulting fragments alloyed and the resulting fragment transform cells of E. coli strain JM109. The obtained plasmid pK0 size 2.8, etc., ad resistant to kanamycin. Identification produced by lengths restricted fragments SalI - HindIII.

Plasmid pK0 hydrolyzing the restriction enzyme AvaII so that one act of hydrolysis accounted for an average of one molecule plasmids. After phenol deproteinization obtained hydrolyzate is treated with nucleases Mung bean. After phenol deproteinization hydrolyzed alloyed with synthetic SalI-linker (5'-GGTCGACC-3') and after inactivation of DNA ligase hydrolyzing the restriction enzyme SalI. After phenol deproteinization mixture is treated with DNA ligase of phage T4 and transform cells of E. coli strain JM109. Among recombinants resistant to kanamycin, select a clone having a size of 2.5 T. p. N. and containing a unique area of recognition of restrictase SalI and receive plasmid pK1.

Example 3. Getting the producer strain.

Cells of Escherichia coli JM109 transformed with two plasmids: pR4 and pUABC22 and get producing strains modified plasminogen activator urokinase type.

Example 4. The preparation of recombinant modified plasminogen activator urokinase type.

Bacteria E. coli strain VKPM B-766 containing plasmids pUABC22 and pR4, grown to a density OD600= 2.5 V for 16 h in 50 ml LB-medium with ampicillin (100 μg/ml) at 37oC and intensive aeration. After cultivation, the cells are harvested by centrifugation for 30 min at 40 is dobavlaut Tris-HCl, pH 8.3 50 mm EDTA to 20 mm, incubated for 30 min at 4oC, and then treated with ultrasound for 1 minute, Sedimentary fraction of cell extract was separated by centrifugation for 90 min at 5000 rpm and suspended in 1 ml of 2% Triton X-100, centrifuged 5 min at 14000 rpm and suspended in 100 μl of 200 mm EDTA, 500 mm Tris-HCl, pH 8.0, add 800 μl of denaturing solution (6 M guanidinium, 100 mm 2-mercaptoethanol), incubated for 20 min at 65oC insoluble precipitate is removed by centrifugation.

The procedure of reconstitute enzymatic activity carried out as follows. The solution was diluted 20-fold with buffer: 100 mm Tris-HCl, pH 9.0, 200 mm arginine-chloride, 5 mm restored glutathione, 1 mm oxidized glutathione, denaturing agent (1 M guanidinium), incubated for 40 h at 4oC, then cialiswhat against two changes of 1 l of 100 mm NaCl, 20 mm Tris-HCl, pH 8.0, within 6 hours of Precipitated precipitate is removed by centrifugation and measured activity in the test for fibrinolysis as described above. Activity Maputo in the sample is 1500.

Denaturirovannyj recombinant modified plasminogen activator presents mainly preferments form with a molecular mass of 43 kDa that demonstrate damassy, that's 4 times the output Maputo, obtained under similar conditions in the case of the producer strain ITCM IN-4534.

1. Recombinant plasmid DNA pUABC22 encoding a modified plasminogen activator urokinase type, having a size of 4.5 T. p. N. and containing XmnI - BamHI - fragment DNA plasmids pUABC22 size 2,99, etc., ad; - BamHI - kpni restriction sites - DNA fragment size 0,177, etc., ad, containing the site of the coding region of the gene Maputo, corresponding to 12 C-terminal amino acids Maputo, and noncoding element - artificial intergenic sequence IHL 14; Cloned - SacI - DNA fragment size of 0.57, etc., ad, containing the gene for tRNA Arg; SacI - XmnI fragment DNA vector pUC19 size of 0.79, etc., B.C., site of replication initiation and promotora-operator region of the lac operon; genetic markers; gene for resistance to ampicillin.

2. Noncoding DNA element - artificial intergenic sequence IHL 14 size of 0.13, etc., H.: -5'-GGGCTGCAGGCCGGGCGGCGCCGTGCGCGCCCGGCCGCCGATGCTCCCCGGCGGCCGGGCGCGCACGGCGCCGCCGGGCACTGCAGCCCCGCGGGGCCCCTCAGGGGGCCCCGCGGGGTTTTTTGGTACC-3'.

3. The bacterial strain Escherichia coli VKPM B-7666 producing a modified plasminogen activator urokinase type.

 

Same patents:

The invention relates to biotechnology and allows you to get the polypeptides used as plasminogen activators having high specific amylolyticus and fibrinolytic activity

The invention relates to the production of tissue plasminogen activator (enhanced APT) with prolonged biological half-life existence, increased resistance to heat and acids and effective as an inhibitor of inflammation around the site where the formed thrombus

The invention relates to Enzymology, particularly to a method of increasing the stability of the enzyme urokinase to heat, and can be used for inactivation of viruses present in the drug

The invention relates to the feeding of material into the cells of the body, namely, the supply of genetic material to living tissue

The invention relates to methods of introducing foreign genetic material into bacteria using vectors in particular to methods of introducing foreign genes into the genomes of gram-negative microorganisms

The invention relates to biotechnology and allows you to get the polypeptides used as plasminogen activators having high specific amylolyticus and fibrinolytic activity

The invention relates to the production of tissue plasminogen activator (enhanced APT) with prolonged biological half-life existence, increased resistance to heat and acids and effective as an inhibitor of inflammation around the site where the formed thrombus

The invention relates to genetic engineering, in particular the production of plasminogen activator in cells E

The invention relates to the preparation of the interest of the enzymes in the seeds of transgenic plants and application of the thus prepared seed and production processes in the absence of the required extraction or isolation of the enzyme

The invention relates to biotechnology, in particular genetic engineering, and is a recombinant phage DNA M13polT7, containing the gene for RNA polymerase of phage T7 and strain of phage M13polT7 producing RNA polymerase of phage T7
Up!