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The Soluble Expression Strategy in E.coli and Cell-free Protein Biosynthesis System

Author: ZhangDanPing
Tutor: CaiJin
School: Zhejiang University
Course: Biochemical Engineering
Keywords: hIGF-1 Soluble expression E. coli Rosetta-gami (DE3) Cell-free protein synthesis system Chaperone
CLC: Q78
Type: Master's thesis
Year: 2011
Downloads: 46
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Abstract


This paper in order to improve the amount of soluble expression of the recombinant protein in E. coli and the cell-free protein synthesis system for the purpose. First target protein to hIGF-1 , by selecting the appropriate strategy to increase its amount of soluble expression in E. coli ; followed by EGFP as a model protein , by rich molecular chaperone of E. coli extracts to improve its no amount of the soluble expression of the protein synthesis system . Obtained from a human liver tumor tissue by RT-PCR method , we first target gene hIGF - 1 and construct the expression plasmid pET32 - hIGF - 1 , followed by analysis of the different E. coli host BL21 (DE3 ) , Rosetta ( DE3 ) and Rosetta -gami (DE3) of TrxA-hIGF-1 soluble expression . The results showed that in the Rosetta-gami (DE3) , E. coli host , hIGF-1 fusion protein of the soluble highest expression . Then, the recombinant E. coli Rosetta-gami (DE3) / pET32-hIGF-1 protein expression conditions optimization of the system , the optimal expression conditions , soluble hIGF - 1 fusion protein expression levels reached a maximum 2.06 g / L. Finally after cell crushing, protein purification, enterokinase cutting , releasing mature hIGF-1 concentration reached 0.42 g / L. E. coli cell-free protein synthesis system , this paper into the molecular chaperone plasmid recombinant Escherichia coli to prepare chaperone - rich extract , and use it to build a cell-free protein synthesis system . In the chaperone - rich cell-free protein synthesis system , the amount of target protein, soluble expression of eGFP has been improved significantly. Of this research work will improve the well - established methods of recombinant protein expression in vitro and in vivo soluble .

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CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)
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