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Extract And Analysis Of Cell Wall Protein Of Pichia Pastoris Displaying Heterologous Protein

Posted on:2013-04-07Degree:MasterType:Thesis
Country:ChinaCandidate:X Y ZhouFull Text:PDF
GTID:2230330374475109Subject:Biochemistry and Molecular Biology
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The developing of yeast surface display technology is very rapidly in a large number ofmicroorganisms display systems. This technology has been widely applied in many fields,showing the broad prospects for the development. The cell wall plays a vital role in thefunction of various display systems. The cell wall of yeast plays an extremely important rolein the maintenance of its function, biological metabolism and metabolic transit, andenvironmental stress response. The biological research of yeast cell wall has become a rapidlydeveloping field. It is of great significance for the interpretation of cell morphogenesis toclarify the network structure in the process of cell cycle regulation mechanisms, as well as thedevelopment and application of new antifungal.Cell wall proteins is an important part of the yeast cell wall and closely related to the finestructure of the cell wall and a variety of physiological functions. It is the premise to establisha stable and efficient extraction method of cell wall protein for cell wall proteome of Pichiapastoris and methanol metabolic regulation studies. Covalent binding and non-covalentbinding cell wall proteins in yeast have been applied to the surface display system, howevermajority of the anchoring proteins are exogenous cell wall protein. In order to develop newendogenous cell wall proteins to make it more suitable for Pichia pastoris to display foreignproteins, we have made the following research:(1) We take use of recombination cell wall Flo1p flocculation of non-covalent bondingyeast GS115/KFS-CALB, GPI (glycosyl phosPhatidylinositol)-covalent bond α lectin yeastGS115/KNS-CALB, PIR (proteins tinternal repeats) covalent bond Pir1protein yeastGS115/PIR1-CALB, with four different methods of hot SDS, Laminarinase, HF-pyridinehydrolysis and mild alkali extracting CWPs of recombinant Pichia pastoris. The resultsindicate that3types of anchored integrated cell wall proteins have been demonstrateddisplaying on the cell wall of Pichia pastoris successfully. It does little effect on cell growthconditions, however there is a big influence on the display of enzyme expression if Pichiapastoris is with three types of bonding anchor proteins to display the lipase. The hydrolaseactivity of lipase of GS115/KFS-CALB, GS115/KNS-CALB and GS115/PIR1-CALB arerespectively up to855.02,1239.40and210.47(per gram dry weight of cells). The CWPsFlo1p flocculation of non-covalent bonding yeast GS115/KFS-CALB would be released byhot SDS, CWPs α lectin of GPI-covalent bonding yeast GS115/KNS-CALB would bereleased by Laminarinase or HF-pyridine, and cell wall Pir1proteins of PIR-covalent bonding yeast GS115/PIR1-CALB would be released by Laminarinase or mild alkali.(2) All proteins of Pichia pastoris are predicted by software of Big-PI Fungal Predictorand the prediction of signal peptide, we found50potential GPI cell wall proteins and madeantarctic candida lipase B a reporter molecular to contribute a display system of Pichiapastoris. With the detection of flow cytometry and Western Blot analysis of cell wallprotein extracted by Laminarinase detected, the results show that there are13fusion proteinsuccessfully displaying on the cell wall, and in line with the characteristics of the GPI protein,proving that these13proteins are GPI cell wall proteins of Pichia pastoris. In addition, it isefficient for the new13GPI cell wall proteins to display CALB, and with high enzymaticactivity of hydrolysis of CALB, however it is different from various cell proteins. Then weanalyze the characteristic of13novel GPI cell wall proteins through bioinformatics software.
Keywords/Search Tags:cell-surface display, Pichia pastors, cell wall protein, extraction method, GPIprotein
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