JOURNAL OF SHANDONG UNIVERSITY(NATURAL SCIENCE) ›› 2015, Vol. 50 ›› Issue (11): 8-15.doi: 10.6040/j.issn.1671-9352.0.2014.573

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Expression and purification of human proinsulin gene in Escherichia coli

ZHANG Xiao-wei1, PENG Zhen-ying1, CHEN Gao1, ZHENG Ling1, YU Jin-hui2, BI Yu-ping1,3, BIAN Fei1   

  1. 1. Biotechnology Research Center, Shandong Academy of Agricultural Sciences, Jinan 250100, Shandong, China;
    2. Crop Germplasm Resources Center, Shandong Academy of Agricultural Sciences, Jinan 250100, Shandong, China;
    3. Graduate Education Center, Shandong Academy of Agricultural Sciences, Jinan 250100, Shandong, China
  • Received:2014-12-22 Revised:2015-02-11 Online:2015-11-20 Published:2015-12-09

Abstract: The human proinsulin (PI) gene sequence was optimized using the Escherichia coli preferred codons. The reformed PI gene was amplified, the fused expression vector (TrxA-PI) was constructed and transformed into three E.coli expression strains BL21(DE3),TransB(DE3) and Rosetta-gami(DE3). SDS-PAGE analysis showed that the expression level of the fused protein was the highest in Rosetta-gami (DE3) strain, which was about 10 times higher than in BL21(DE3) strain. The production of soluble fused protein was 3.5 mg/mL after optimization of the fermentation conditions. The purified recombinant PI was obtained after enterokinase digestion and HisTrap FF column purification, and its antigen activity was measured using Western-blot analysis.

Key words: proinsulin, E.coli expression, preferred codons

CLC Number: 

  • Q815
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