Holmquist M, Tessier D C, Cygler M
Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec, Canada.
Protein Expr Purif. 1997 Oct;11(1):35-40. doi: 10.1006/prep.1997.0747.
We describe the heterologous high-level expression of the two Geotrichum candidum lipase (GCL) isoenzymes from strain ATCC 34614 in the methylotrophic yeast Pichia pastoris. The lipase cDNAs were placed under the control of the methanol-inducible alcohol oxidase promoter. The lipases expressed in P. pastoris were fused to the alpha-factor secretion signal peptide of Saccharomyces cerevisiae and were secreted into the culture medium. Cultures of P. pastoris expressing lipase accumulated active recombinant enzyme in the supernatant to levels of approximately 60 mg/L virtually free from contaminating proteins. This yield exceeds that previously reported with S. cerevisiae by a factor of more than 60. Recombinant GCL I and GCL II had molecular masses of approximately 63 and approximately 66 kDa, respectively, as determined by SDS-PAGE. The result of endoglucosidase H digestion followed by Western blot analysis of the lipases suggested that the enzymes expressed in P. pastoris received N-linked high-mannose-type glycosylation to an extent, 6-8% (w/w), similar to that in G. candidum. The specific activities and substrate specificities of both recombinant lipases were determined and were found to agree with what has been reported for the enzymes isolated from the native source.
我们描述了来自菌株ATCC 34614的两种白地霉脂肪酶（GCL）同工酶在甲基营养型酵母毕赤酵母中的异源高水平表达。脂肪酶cDNA置于甲醇诱导型醇氧化酶启动子的控制之下。在毕赤酵母中表达的脂肪酶与酿酒酵母的α-因子分泌信号肽融合，并分泌到培养基中。表达脂肪酶的毕赤酵母培养物在上清液中积累了活性重组酶，其水平约为60 mg/L，几乎不含污染蛋白。该产量比先前报道的酿酒酵母产量高出60多倍。通过SDS-PAGE测定，重组GCL I和GCL II的分子量分别约为63 kDa和约66 kDa。对脂肪酶进行内切葡糖苷酶H消化后再进行蛋白质印迹分析的结果表明，在毕赤酵母中表达的酶接受了N-连接的高甘露糖型糖基化，其程度为6-8%（w/w），与白地霉中的情况相似。测定了两种重组脂肪酶的比活性和底物特异性，发现与从天然来源分离的酶的报道一致。
