The Comparison And Analysis Of Calb Display Capability In Pichia Display System

The yeast pichia pastoris is a useful eucaryon expressing system which has developed in these yeasrs. This system has several good quality, for example, high amouts expression, clutivating low, product can secrete outside,proper glycosylation. At present, P.pastoris has been studied for protein-surface display based on the anchor system of the flocculation functional domain of Follp(FS) from S.cerevisiae and the C domain of GPI anchor system which were noncovalently and covalently linked to the cell wall respectively.On the basis of previously work, laboratory constructed the gene of CALB which was displayed on the pichia pastoris using three different anchor systems.On this basis,the article is divided into two parts.1.Comparing the display and secreion ability of foreign protein in different pichia pastoris display system.Western blot confirmed that the fusion proteins KFS-CALB/GS115 were released from cell walls after mild alkaline treatment,and can excrete into fermentation supernatant abundantly, conversely,the proteins KNS-CALB/GS115 and Sedl-CALB/GS115 can only extracted using glucanase with less secretory capacity. No matter which anchor system, the practical molecular weight of interest protein is more larger than its in theory which maybe is associated with glycosylation of fusion protein. The fluorescence intensity of the whole yeast was measured by flow cytometry, in which we found that three yeast strain with different anchor system had different fluorescence intensity that KFS had maximum and Sedl was smallest. The result indicate that the anchor efficiency was variance obviously with different anchor system even in the same host and the same foreign protein. To analyse the hydrolyse activity and protein concentration of fermentation supernatant we found that the high secretion capability of KFS was related to its noncovalent bonding possibility. Moreover, the micromolecule band in superanant of KFS showed that the excreted protein was associated with protease probablely.2.The analyse of CALB in pichia display system with KFS anchor system. By the experiment of mono-factor,the main results as follows:under 28℃the expression of the protein is the highest. and 2% peptone was added to culture medium is the best, at the same time, Additions of Gly, Ala, Pro into the medium,0.1% concentrationhas the optimization expression. Neverthelessly, the serection level was increasing along with the raising of display ability. The 2DGE of the supernatant of KFS pichia pastoris and GS115 showed that the protein were concentrated in the acidity terminal and we can see the fusion protein in the macromolecule place.The study analyze the display capability of pichia pastoris display system further, in which we found the KFS recombinant yeast that incorporated with flocculation anchor system had the biggest expression ability of the fusion lipase,either in the surface of cell or the fermentation supernate,and had the highest plasticity.lt had a big influence by the expression of fusion protein to change cultivation conditon.