High-level Expression And Functional Identification Of Human Epidermal Growth Factor In E.coli

Human Epidermal Growth Factor(hEGF)is a kind of active substances in human body.hEGF not only can stimulate cell proliferation,differentiation and migration,but also plays an extremely important role in wound healing,organ generation and cell signal transduction,therefore hEGF has been widely used in medicine and cosmetics.With more and more attention to hEGF,the research on biosynthesis of hEGF has never been stopped.However,hEGF exists three intra-molecular disulfide bonds,which is why it is difficult to soluble expression in prokaryotic expression system.In this paper hEGF was expressed as a soluble way in E.coli by the experiment with different expression strategy and optimizing the conditions,meanwhile the activity of hEGF is identified by the method of methylthiazoletetrazolium.In this study,hEGF gene was fused with different genes or co-expressed with molecular chaperone to achieve the soluble expression of hEGF in E.coli,and also hEGF was active after purification.Detailed work were done as following.Firstly,hEGF gene was fused with SUMO protein tags and it was found to facilitate soluble expression,and the fusion protein coexpressed with molecular chaperone further improved hEGF soluable production,natural hEGF can be obtained after the SUMO protein tag was cleaved by SUMO protease.Secondly,Cterminal cleavage intein was inserted between the SUMO gene.Thirdly,SUMO gene was fused to the C-terminal of hEGF and inserted N-terminal cleavage intein to solve the self-cleavage problem of premature protein of C-terminal cleavage intein in vivo.The major results are as follows.While The recombinant plasmid His-SUMO-hEGF(HSE)respectively was co-expressed with different molecular chaperone proteins(pG-KJE8,pKJE7,pGro7,pG-Tf2,pTf16),pKJE7 had the most powerful effect on soluable expression.The most optimal expression conditions included TB culture medium,0.2 mg/ml of L-Arabinose for molecular chaperone protein expression,0.5mM IPTG for target protein,25 ?,180 rpm,24 hours.The production of His-SUMO-hEGF is 78 mg/L,The final yield of bioactive hEGF after further purification by the removal of SUMO tag is about 20.6 mg/L,increased by 28.75% compared with no molecular chaperone co-expression.The recombinant plasmids His-SUMO-ssp dnaB-hEGF(HSSE)and the SUMO-ssp dnaBhEGF-His(SSEH)were transformed into BL21(DE3).The most optimal expression conditions were as follows: TB culture medium,25?,180 rpm and 24 hours.The fusion proteins were observed to be self-cleavage before exhaustive prematuration,hEGF and hEGF-His failed to be obtained finally.The recombinant plasmid hEGF-Mxe GyrA-SUMO-His(EMSH)was transformed into BL21(DE3).The most optimal expression conditions were as follows: TB culture medium,25? and 24 hours.The intein would occur N-terminal cleavage after 20 mM DTT was added to the buffer,the final production of bioactive hEGF is 29.4 mg/L.This study significantly improved the yield and reduced the cost of hEGF expressed in E.coli in vivo system.We systematically studied the sysnthesis strategy to express recombinant protein containing intramolecular disulfide bonds in reduction system.This paper with scientific significance and social benefits provided the theoretical basis and new ideas to produce efficiently complex eukaryotic proteins in prokaryotic expression system.