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Method for producing recombined streptokinase

A streptokinase, purpose technology, applied in the production field of recombinant streptokinase, can solve the problems of high product cost, complex production process, low yield and the like

Inactive Publication Date: 2006-07-05
SHANGHAI NEWSUMMIT BIOPHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Currently, the recombinant streptokinase on the market adopts the expression system of Escherichia coli, and the expression product is an insoluble inclusion body. Therefore, it needs denaturation and renaturation to obtain an active product. The production process is complicated, the yield is low, and the product cost is relatively high.

Method used

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  • Method for producing recombined streptokinase
  • Method for producing recombined streptokinase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Construction of r-SK secretion expression system

[0067] 1. Obtaining the target gene

[0068] The expression plasmid is a pXSY expression vector (the expression vector not only contains an arabinose-induced promoter but also has the function of guiding the target protein to the intercellular substance), and the host bacterium is Escherichia coli.

[0069] According to the natural amino acid sequence of r-SK reported in the literature, according to the codon preference of Escherichia coli, under the condition of not changing the amino acid sequence, the whole sequence of the recombinant streptokinase (r-SK) gene was artificially synthesized, and at the same time, the 5' of the gene The NcoI site consistent with the new source expression vector was introduced at the end and the EcoR I site was introduced at the 3' end. The artificial synthesis of the gene was completed by Shanghai Sangon Bioengineering Technology Co., Ltd.

[0070] 2. Construction of expression plasmid...

Embodiment 2

[0074] Medium selection

[0075] Element

[0076] Results: The amount of glycerol had no significant effect on the expression level, but too little phosphate was unfavorable to the growth of the bacteria, and the growth of the bacteria in medium 1 and 2 was slow. Among all media, No. 4 had the best effect.

Embodiment 3

[0078] Express on the tank (5L)

[0079] 1. Strains:

[0080] The Escherichia coli containing the pXinogen / SK expression plasmid was cultivated in LBA for 20hr, OD 600 up to 2.7.

[0081] Two, basal medium: medium 4 in embodiment 2

[0082] 3. Fermentation and cultivation stage:

[0083] Temperature: 37°C

[0084] pH: 7.0

[0085] OD 600 : 3.0

[0086] Co-culture for 5.5 hours

[0087] 4. Fermentation induction stage:

[0088] Temperature: 35°C

[0089] pH: 6.8

[0090] L-arabinose: lactose addition amount: 3:1 (quantity ratio, wherein the concentration of L-arabinose is 0.2%)

[0091] end of induction OD 600 : 8.8

[0092] Total induction 2 hours

[0093] 5. Experimental results:

[0094] After 2 hours of induction, r-SK expression accounted for about 50% of the total protein in the fermentation supernatant, and the expression amount reached 1000 mg / L fermentation broth (measured by the improved Lowry method), and the protein activity was about 8 × 10 7 IU / L f...

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Abstract

This invention describes a process for producing a streptokinase r-SK, comprising the steps of: a) under appropriate expression conditions, cultivating colibacillus engineering cells that carry an expression carrier selected from the following group: the carrier that contains arabinose-induced promoters, or the carrier that not only contains arabinose-induced promoters but also possess the function of directing the targeting proteins to interstitial cells. The coding sequence of the streptokinase is inserted into the expression carrier in order to express soluble and highly active r-SK; b) separating and purifying the step a) wherein said r-SK. In the process of this invention, the expression amount is high, and the separation and purification steps are simple. This invention also describes corresponding expression carriers and engineering cells.

Description

technical field [0001] The invention relates to a production method of recombinant streptokinase (Recombinant Streptokinase), as well as expression vectors and host cells used in the method. Background technique [0002] Under normal physiological conditions, the coagulation system, anticoagulation system and fibrinolytic system in the blood maintain a dynamic balance, which not only ensures the potential coagulability of the blood but also ensures the fluid state of the blood at all times. But in some pathological conditions, the above balance will be broken. Damage to the vascular endothelium may occur in some parts of the body, and these parts may undergo a coagulation process on the basis of platelet aggregation to form small thrombi. [0003] The anticoagulant factors in the blood play an important role in the anticoagulant function. There are three main systems: thrombin III, protein C system and tissue factor pathway inhibitor (TFPI). What play an important role in ...

Claims

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Application Information

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IPC IPC(8): C12N9/70C12N15/09C12N15/70
Inventor 黄阳滨胡萍丰涛
Owner SHANGHAI NEWSUMMIT BIOPHARMA
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