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Method and use of producing soluble recombinant protein in colibacillus

A technology of Escherichia coli and protein, applied in the field of genetic engineering biology, can solve the problems of time-consuming, laborious, difficult separation and purification and quality control in the production process, and achieve the effect of increasing production, simple method, and inhibiting the proliferation of vascular endothelial cells

Active Publication Date: 2007-08-29
NANJING JIRUIKANG BIOTECHNOLOGY RES INST CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, recombinant human endostatin in clinical research in China is mostly produced by in vitro refolding of E. coli inclusion body products. The production process is time-consuming, laborious, and difficult to separate, purify, and quality control.

Method used

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  • Method and use of producing soluble recombinant protein in colibacillus
  • Method and use of producing soluble recombinant protein in colibacillus
  • Method and use of producing soluble recombinant protein in colibacillus

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1: Cloning, expression, isolation and purification and characterization of recombinant human endostatin gene:

[0035]1. Cloning of human endostatin cDNA and construction of expression plasmid: Grind 50 mg of human liver tissue, dissolve it in 1ml TRIZOL RNA Isolation Reagent (Gibco BRL), isolate RNA according to the instructions of the reagent, and use the isolated RNA as a template. One-step RT-RNA, cDNA amplification, the method is carried out according to the instructions of OneStep RT-RNA PCR Kit (TaKaRa), the forward primer used: 5'-ttc cat atg cacagc cac cgc gac ttc cag-3', which adds Nde I enzyme Cut site (see the bold part); reverse primer 5'-ccg ctc gag cta ctt gga ggc agt cat g-3', a translation stop codon (CTA) is added after the endostatin coding sequence, for cloning convenience, The restriction site XhoI (in bold) was also added. The obtained PCR product was about 550bp. The obtained PCR product and plasmid pET23a (Novagen) were digested with Nde I an...

Embodiment 2

[0042] Example 2: Synthesis, cloning, expression, isolation and purification and characterization of recombinant human angiostatin gene:

[0043] 1. The synthesis and cloning of human angiostatin gene and the construction of its expression vector: referring to the code word preferred by E. coli, ten oligonucleotides were chemically synthesized, the length of the oligonucleotides is 28-35 bases, adjacent The oligonucleotides have 5-8 bases paired with each other. See the figure for the sequence of the oligonucleotides and the overlap pattern between them. First, dilute all oligonucleotides to 100pmol / μl, and then follow the following steps in sequence: T4 polynucleotide kinase phosphorylation, 30°C annealing, Klenow large fragment fills in the gap, T4 DNA ligase connects all the gaps (nick), finally got a 183bp double-stranded DNA fragment encoding human calreticulin from amino acids 120 to 180. The fragment was amplified by PCR, the forward primer used was: 5’-CGGGATCCTGGACGACGACG...

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Abstract

The invention was involved in that target protein expression strain and molecular partner were expressed in E.coli.at the same time at low temperature to prevent the formation of target protein inclusion body effectively and increase yields of soluble production largely. Soluble recombinant human endostatin and human angiostatin were increased largely by the technique. It provided a new simple and cheap technique to produce soluble recombinant protein with biological activity. It was applied in bioengineering pharmaceutical factory, gene engineering, biochemistry and molecular biology with high efficiency, cheap price, wide application and strong promotion.

Description

1. Technical Field: [0001] The invention belongs to the field of genetic engineering biotechnology. 2. Background technology: [0002] The E. coli expression system has the characteristics of fast growth, clear genetic background, high expression level and low cost. It is an ideal foreign protein expression system. However, the biggest disadvantage of this expression system is that foreign proteins often fail to fold correctly and exist in the form of inactive inclusion bodies, and the yield of inclusion body refolding is usually very low. Finding simple, easy and versatile methods to improve or prevent the formation of inclusion bodies is a difficulty and bottleneck in the current E. coli expression system. [0003] Angiogenesis is necessary for the growth and metastasis of solid tumors. In order to promote angiogenesis, tumor tissues produce a series of angiogenic factors (such as bFGF), and many tumors also produce anti-angiogenic proteins, one of which is endostatin (endostat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N1/21C12P21/02C12R1/19
Inventor 华子春徐寒梅孙启明
Owner NANJING JIRUIKANG BIOTECHNOLOGY RES INST CO LTD
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