重组毕赤酵母细胞壁蛋白的抽提和分析

Extraction and Analysis of Cell Wall Proteins of Recombinant Pichia pastoris

建立稳定有效的细胞壁蛋白(CWPs)抽提方法,是开展毕赤酵母细胞壁蛋白质组学及甲醇代谢调控相关性研究的基础.为此,以具重组非共价结合壁蛋白Flo1p絮凝素的酵母GS115/KFS-CALB、具共价结合壁蛋白α凝集素的酵母GS115/KNS-CALB和具重组共价结合壁蛋白Pir1的酵母GS115/Pir1-CALB为对象,用热十二烷基磺酸钠(SDS)、昆布多糖酶、氢氟酸吡啶和温和碱水解对上述壁蛋白进行抽提,结果表明:3种壁蛋白已成功地展示于毕赤酵母细胞壁的表面;用这3种毕赤酵母锚定蛋白展示脂肪酶,对细胞的生长状况影响不大,而对展示酶的表达量影响较大,GS115/KFS-CALB、GS115/KNS-CALB和GS115/Pir1-CALB的脂肪酶水解比酶活最高达855.02、1239.40和210.47 U/g(以干细胞计);热SDS可有效抽提GS115/KFS-CALB上的Flo1p絮凝素,昆布多糖酶和氢氟酸吡啶可释放GS115/KNS-CALB上的α凝集素,而昆布多糖酶和温和碱可释放GS115/Pir1-CALB上的Pir1.

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/KNS-CALB 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.