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Intein Mediated Expression Of Cyclic Sunflower Trypsin Inhibitor-1 In E. Coli

Posted on:2011-01-15Degree:MasterType:Thesis
Country:ChinaCandidate:W ZhangFull Text:PDF
GTID:2120360302980167Subject:Biochemistry and Molecular Biology
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The sunflower trypsin inhibitor-1(SFTI-1) is a recently discovered cyclic inhibitor containing 14 amino acid residues.It is the smallest and the most potent Bowman-Birk inhibitor.It has been shown that SFTI-1 also has inhibitory action on rennin and elastase.Because of its small size and strong activity,SFTI-1 is a very attractive template for the design and chemical synthesis of new proteinase inhibitors as potential therapeutic agents.More and more people are paying attention to the cyclic skeleton of SFTI-1 with potent trypsin inhibitory activity,and are trying to synthesize SFTI-1 by different ways.The traditional ways of solid or liquid phase peptide synthesis are expensive and laborious for synthesizing SFTI-1.Here,we prepared the cyclic SFTI-1 by intein mediated expression in E.coli BL21 using pTwin1 vector.The expression conditions were found to be 37℃,4 hours, dissolved oxygen 35%and IPTG concentration 0.1 mM.A fusion protein with linearized SFTI-1 sandwiched between two inteins was expressed and purified by chitin affinity chromatography. After the specific self splicing of N-terminal and C-terminal inteins,the N- and C-terminal residues of linear SFTI-1 were joined spontaneously to form cyclic SFTI-1.The product was further purified by trypsin affinity chromatography.Mass spectrometry confirmed that the molecular weight of synthetic SFTI-1 was consistent with the theoretical value.The yield of SFTI-1 was about 2.8 mg per liter of fermentation culture.
Keywords/Search Tags:sunflower trypsin inhibitor-1 (SFTI-1), cyclic peptide, intein mediated expression
PDF Full Text Request
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