Molecular Cloning and Expression of Gene Coding for the Analog of Trichosanthes Trypsin Inhibitor in E.coli and Saccharomyces cerevisiae  

作　　者：陈小明 钱岳伟 戚正武 

机构地区：[1]National Molecular Biology Laboratory,Shanghai Institute of Biochemistry,Academia Sinica,Shanghai 200031,PRC

出　　处：《Progress in Natural Science:Materials International》1993年第2期144-154,共11页自然科学进展·国际材料（英文版）

基　　金：Project supported by the State Biological High Technology Research Grant of China.

摘　　要：The gene coding for the analog of Trichosanthes trypsin inhibitor((Ala-6)-TTI)was cloned inexpression vector pWR590-1.The expressed fusion protein was cleaved with cynogen bromide and reduced.After refolding with trypsin Sepharose as a matrix and affinity chromatography on the immobilized enzyme,the fully active(Ala-6)-TTI was obtained.The trypsin inhibitory activity of the refolded(Ala-6)-TTI was con-sistent with the natural one,and its amino acid composition agreed well with the predicted value.To makethe reading frame of TTI gene compatible with the secreted expression vector in S.cerevisiae,the(Ala-6)-TTIgene was inserted with a nucleotide via the site-directed mutagenesis,and then cloned into the expressionvector pVT102U/α to transform S.cerevisiae S-78.The trypsin inhibitory activity was clearly found in thesupernatant of the culture.The secreted(Ala-6)-TTI was purified and found to be correctly processed at thejunction between the α-factor leader peptide and the(Ala-6)-TTI downstream.Comparing the two expressionsystems,i.e.E.coli and S.cerevisiae,the latter has a greater advantage for high yield(】2 mg/1)and easypurification of the expressed product without disulfide refolding.The gene coding for the analog of Trichosanthes trypsin inhibitor((Ala-6)-TTI)was cloned in expression vector pWR590-1.The expressed fusion protein was cleaved with cynogen bromide and reduced. After refolding with trypsin Sepharose as a matrix and affinity chromatography on the immobilized enzyme, the fully active(Ala-6)-TTI was obtained.The trypsin inhibitory activity of the refolded(Ala-6)-TTI was con- sistent with the natural one,and its amino acid composition agreed well with the predicted value.To make the reading frame of TTI gene compatible with the secreted expression vector in S.cerevisiae,the(Ala-6)-TTI gene was inserted with a nucleotide via the site-directed mutagenesis,and then cloned into the expression vector pVT102U/α to transform S.cerevisiae S-78.The trypsin inhibitory activity was clearly found in the supernatant of the culture.The secreted(Ala-6)-TTI was purified and found to be correctly processed at the junction between the α-factor leader peptide and the(Ala-6)-TTI downstream.Comparing the two expression systems,i.e.E.coli and S.cerevisiae,the latter has a greater advantage for high yield(>2 mg/1)and easy purification of the expressed product without disulfide refolding.

关 键 词：SQUASH family PROTEINASE inhibitor Tricho■es kirilo■ reco■t DNA SITE-DIRECTED ■genesis fu■ion protein EXPRESSION in E.COLI gene EXPRESSION and secretion in SACCHAROMYCES cerevisiae 

分 类 号：N[自然科学总论]