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The Cell Surface Display-releasing System, Construction And Application

Posted on:2012-12-13Degree:MasterType:Thesis
Country:ChinaCandidate:W Y GaoFull Text:PDF
GTID:2120330335950406Subject:Biochemistry and Molecular Biology
Abstract/Summary:PDF Full Text Request
Endoglucanase played a key role in the process of cellulose synergistic degradation. Directed evolution was an important way to obtain high activity endoglucanase. It was a challenge to make free access of the mutant to insoluble substrate extracellularly for the directed evolution of Endoglucanase. As a result, In this paper we designed a new type of cell surface display-releasing system based on cell surface display system. The carrier protein is ice nucleation protein (INP), We embedded intein between cellulose and INP. The cellulase was released from the cell surface that used intein function of self-shearing to achieve the freely interaction between mutant and the insoluble cellulose. The optimal conditions of protein self-releasing was investigated using a red fluorescent protein (RFP) as a reporter. The results showed that in 20mM pH 7.9 Tris-HCl+0.9% NaCl lysis buffer,37℃,48h, the red fluorescent protein could release from E. coli cell surface release, the release efficiency could reach about 50%, adding an appropriate amount of reducing agent, cleavage efficiency would be improved.Based on these studies, according to the principle of intein shearing, we designed further the cell surface display-controlled releasing system to improve the carrying capacity, and the purposes for controlled releasing. The optimal conditions of protein self-releasing was investigated using a red fluorescent protein (RFP) as a reporter. in 20mM pH 7.9 Tris-HCl+0.9% NaCl lysis buffer,37℃, the addition C-terminal that N-terminal complementary fragment and 10mM DTT, the red fluorescent protein from E. coli cell surface was released, its efficiency could reach 40-50%.We used protein engineering technology to cloning and expression endoglucanase CpCe15C from Clostridium phytofermentans, and CpCe15C was built into the system (pET20bp-INP-I-Ce15C and pET20b INP-IN-Ce15C), respectively, determined cell surface display and release function, the results showed that both cell surface display-releasing system can released cellulase to the extracellular which could maintain its vitality.In this article, two novel cell surface display-release systems were built. They would be an efficient platform used for directed evolution of cellulase,and they also showed promising potentiality for broadly use in biology research.
Keywords/Search Tags:cell surface display, Ice nucleation protein, intein, self-splitting, CpCel5C
PDF Full Text Request
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