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Preparation method of micro-plasmin without self-cutting form

A technology of fibrinolytic enzymes and forms, applied in the field of enzyme preparation, to reduce the probability of self-degradation, improve market competitiveness, and reduce the use of

Active Publication Date: 2019-07-30
CHONGQING PEG BIO BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] The present invention provides a method for preparing microplasmin without self-cutting, which solves the problem of not only avoiding the stability of microplasmin due to the acid hydrolysis factor of too low pH in the prior art, but also avoiding the problem caused by too high pH value. The problem of autocatalytic degradation of microplasmin

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  • Preparation method of micro-plasmin without self-cutting form
  • Preparation method of micro-plasmin without self-cutting form
  • Preparation method of micro-plasmin without self-cutting form

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Embodiment 2

[0065] Embodiment 2: adopt all steps in above-mentioned seed liquid culture and liquid fermentation culture, obtain fermented liquid (wherein, keep in step S1232 (NH 4 ) 2 SO 4 The concentration is 0.1mol / L and sucrose induction for 24h), and then the fermentation broth is subjected to electrophoresis to detect the expression level of the target protein. During electrophoresis, the volume ratio of the fermentation broth to the polyacrylamide gel is 85%:15%.

Embodiment 3

[0066] Embodiment 3: adopt all steps in above-mentioned seed liquid culture and liquid fermentation culture, obtain fermented liquid (wherein, keep in step S1232 (NH 4 ) 2 SO 4 The concentration is 0.25mol / L and sucrose induction for 24h), and then the fermentation broth is subjected to electrophoresis to detect the expression level of the target protein. During electrophoresis, the volume ratio of the fermentation broth to the polyacrylamide gel is 85%:15%.

Embodiment 4

[0067] Embodiment 4: adopt above-mentioned seed liquid culture and all steps in liquid fermentation culture, obtain fermented liquid (wherein, in step S1232 keep (NH ) SO Concentration is 0.5mol / L and sucrose induction 24h), then this fermented liquid is electrophoresed To detect the expression level of the target protein, the volume ratio of the fermentation broth to the polyacrylamide gel during electrophoresis is 85%:15%.

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Abstract

The invention provides a preparation method of micro-plasmin without a self-cutting form. The preparation method comprises the steps of S1, recombinant fermentation of the micro-plasmin without the self-cutting form, wherein recombinant fermentation comprises the steps of S12, liquid fermentation culture, namely (NH4)2SO4 and sucrose are added in the liquid fermentation culture process. By the adoption of the preparation method of the micro-plasmin without the self-cutting form, the problems are solved that in the prior art, micro-plasmin acid hydrolysis caused by excessively low pH and self-catalytic degradation of the micro-plasmin caused by excessively high pH cannot be avoided at the same time.

Description

technical field [0001] The invention relates to an enzyme preparation method, in particular to a method for preparing microplasmin without self-cleavage. Background technique [0002] Fibrinolytic enzyme (plasmin) is a serine protease derived from plasminogen, an important constituent of mammalian blood. Human plasminogen (Plasminogen, Plg) is a multi-domain protein consisting of 791 amino acid residues, consisting of an N-terminal proactivation domain, five homologous Kringle domains (each about 80 amino acids), a The catalytic domain of serine protease and the connection sequence between domains, with a molecular weight of about 92,000 Daltons, is one of the key components of the human fibrinolytic system. Use plasmin to cut the N-terminal Arg68-Met69 of human plasminogen, or The peptide bond between Lys77-Lys78 or Lys78-Val79 produces a shortened zymogen called lysine-plasminogen. Further cleavage by elastase removes the first 4 Kringle domains to produce another zymoge...

Claims

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Application Information

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IPC IPC(8): C12N9/68
CPCC12N9/6435C12Y304/21007
Inventor 杨辉何勇冷国政黄金凤潘武生范开
Owner CHONGQING PEG BIO BIOTECH CO LTD
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