Abstract:

In order to explore a stable and effective extraction method of cell wall proteins ( CWPs) for the research on the cell wall proteome of Pichia pastoris and the related metabolic regulation of methanol,three kinds of yeasts,namely GS115 /KFS-CALB with recombined non-covalently-bonded cell wall Flo1p protein flocculin,GS115 /KNSCALB with covalently-bonded cell wall α lectin and GS115 /Pir1-CALB with recombined covelently-bonded cell
wall Pir1 protein,were used to extract cell wall proteins using hot SDS,laminarinase,HF-pyridine and mild alkali. The results indicate that the three types of CWPs mentioned above have successfully displayed on the cell wall of Pichia pastoris,that the three tpyes of anchored proteins all have little effect on the cell growth but great effect on the expression of displayed enzyme when they are used to display the lipase,and that the specific hydrolase activity of lipase of GS115 /KFS-CALB,GS115 /KNS-CALB and GS115 /Pir1-CALB are respectively up to 855. 02,1239. 40 and 210. 47 U per gram of dry cells. It is also found that hot SDS is effective in releasing the CWPs Flo1p flocculin of GS115 /KFS-CALB,that laminarinase and HF-pyridine are effective in releasing the CWPs α lectin of GS115 /KNS-CALB,and that both laminarinase and mild alkali are effective in releasing the Pir1 proteins of
GS115 /Pir1-CALB.

Key words: Pichia pastoris, cell wall protein, non-covalent bonding, covalent bonding, extraction method