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The Cell Surface Display-releasing System, Construction and Application

Author: GaoWenYing
Tutor: ZhangZuoMing
School: Jilin University
Course: Biochemistry and Molecular Biology
Keywords: Cell surface display Ice nucleation Protein intron Since the shear CpCel5C
CLC: Q50
Type: Master's thesis
Year: 2011
Downloads: 41
Quote: 0
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Abstract


Played a key role in the process of endo-cellulase cellulase synergistic degradation of cellulose. Directed evolution is an important way to get the high vitality endo cellulase. Endo cellulase directed evolution main difficulty lies in how to achieve the free interaction of the mutant enzyme with extracellular insoluble natural substrate. Based on the above understanding, the papers in the cell surface display system based on the design of a novel cell surface display - release system. Ice nucleation (INP) as a carrier protein, is embedded between the INP and cellulase the intein (Intein), using the Intein self shear function, the cellulase enzyme is released from the cell surface in order to achieve a mutant The enzymes free with water-insoluble cellulose The purpose of the interaction. Red fluorescent protein as the reporter protein, to study the conditions of the automatic release of the results showed that: in the 20 mM Tris-HCl, 0.9% NaCl, pH 7.9 Lysates, 37 ° C, placed under conditions of 48h, the release of a red fluorescent protein from the cell surface. release efficiency of about 50%, adding the right amount of reducing agent, the cleavage efficiency will improve. Further designed on the basis of these studies, based on the Intein shear principle, cell surface display - controlled release system and to improve the carrying capacity of, to achieve a controlled release purposes. Specifically, intein divided 11aa the N-terminal (IN) and 144aa of the C-terminal, the IN connected to the precursor protein, a sandwich structure is formed the INP-in-target protein. The need for N-terminal cleavage occurs when the IC was added to the reaction system, IC can be identified IN, the N-terminus of the catalytic IN NS acyl transfer occurs, an ester bond is hydrolyzed, thereby completing the N-terminal cleavage. Red fluorescent protein as the reporter protein, optimizing the conditions for a controlled automatic release of the target protein, the results showed that: in 20mM pH 7.9 Tris-HCl 0.9% NaCl in lysis buffer, 37 ℃ added under the complementary intein N-terminal fragment of C-terminal and 10mM DTT, red fluorescent protein from the cell surface release efficiency of 30-40%. Cloning and expression of protein engineering techniques from the Clostridium phytofermentans the endo cellulose CpCe15C, and this fiber enzyme built into the two systems (pET20bp-INP-I-Ce15C pET20b INP-IN-Ce15C) cells were measured surface display and release function, the results show that the two cell surface display - release system can be released into the purpose cellobiohydrolase extracellular, and can maintain its original vitality. The paper design build two novel cell surface display - release system, a state-of-the-art research platform will be provided for the directed evolution of cellulase. Will have a very broad use of space biology research.

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