Technology for preparing active t-PA by procaryotic cells

A technology of prokaryotic cells and prokaryotic bacteria, applied in the field of high-efficiency expression, can solve the problems of high product cost, high price and low yield, etc.

Inactive Publication Date: 2008-07-23
王尚武 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The human t-PA currently used clinically, including its mutants, is produced by the eukaryotic CHO system, so it is unavoidable to encounter the production ...

Method used

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  • Technology for preparing active t-PA by procaryotic cells
  • Technology for preparing active t-PA by procaryotic cells
  • Technology for preparing active t-PA by procaryotic cells

Examples

Experimental program
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Embodiment Construction

[0026] 1. Mutation of wild-type human t-PA: the method is to use gene mutation technology to make the amino acid 296-299 in the wild-type protease function P region of wild-type human t-PA combined with plasmin activator inhibitor-1 (Lys-His-Arg-Arg) was mutated to (Ala-Ala-Ala-Ala). And a new endonuclease Not1 site was formed in the mutation region.

[0027] 1). Using wild-type human t-PA as a template, artificially synthesized primers 1, 2, 3 and 4 were used for PCR amplification, respectively.

[0028] Primers were designed as follows:

[0029] Primer 1: (27mer, and introduce Nco1 endonuclease site, beside the character box)

[0030] 5'-GCG gagc cagatcttac caag-3'

[0031] Primer 2: (34mer, and introduce Not1 endonuclease site, beside the character box)

[0032] 5'-ccgctccgggcgat ggcaaagat-3'

[0033] Primer 3: (34mer, and introduce Not1 endonuclease site, beside the character box)

[0034] 5'-ttgcc tcgcccggagagcggtt-3'

[0035] Primer 4: (33mer, and introduce ...

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Abstract

The invention relates to a high-expression prokaryotic cell expression system of non-sugar macromolecule protein containing a plurality of disulfide bonds or protein without important effect on the function affected by glycosyl, in particular to a prokaryotic cell expression system used for expressing a mutant type Plasminogen Activator (mt-PA) with bioactivity, which is characterized in that the mutant type Plasminogen Activator expression sequence (mt-PA) is constructed; an expression plasmid, which comprises an arabinose operon P promoter and the regulatory gene araC and is used for expressing the regulatory elements, is constructed, wherein, the expression plasmid contains the prokaryotic expression vector of the bacteria OmpA secreting signal peptides and the expression of mt-PA; an expression plasmid with isomerism enzyme DsbC or an expression plasmid without signal peptide DsbC is constructed; the expression plasmid of mt-PA and the DsbC or the expression plasmid without signal peptide DsbC are cotransformed to the trxB/gov mutated competent host strain; a secretion type human mt-PA engineering bacteria is constructed, which has the advantages of high level expression and biological activity.

Description

technical field [0001] The invention relates to the construction of the expression structure of human tissue-type plasminogen activator (t-PA, the same below) and its high-efficiency expression in engineered prokaryotic cells. The product has biological activity and can be used for the treatment of thrombotic diseases. The technical field is related to life medicine and biotechnology. Background technique [0002] Human t-PA plays a very important physiological role in the blood coagulation and hemolysis system in the human body. t-PA is coded by 527 amino acids and has 18 disulfide chains in the protein structure. The configuration formed by it plays an extremely important role in the physiological function of the protein. In terms of protein structure, human t-PA is composed of five different functional regions, which are (1). Ring finger functional region; (2). Growth factor functional region; (3). and (4). Two regions with similar functions, Kringle 1 and Kringle2, th...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/58C12N15/66C12N1/21A61K35/74A61P9/10A61P7/02A61P11/00C12N15/70C12N15/74A61K38/16A61K48/00
Inventor 王尚武朱雅南
Owner 王尚武
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