Functional expression of horseradish peroxidase in Saccharomyces cerevisiae and Pichia pastoris
Functional expression of horseradish peroxidase in Saccharomyces cerevisiae and Pichia pastoris
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DOI：
10.1093/protein/13.5.377
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发表时间：
2000-05-01
期刊：
影响因子：
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通讯作者：
Arnold, FH
中科院分区：
文献类型：
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作者：
Morawski, B;Lin, ZL;Arnold, FH
通过定向进化工程化蛋白质的能力需要靶多肽在适合于构建和筛选酶变体文库的重组宿主中的功能性表达。细菌和酵母是优选的，但真核蛋白质通常不能在这些细胞中以活性形式表达。我们试图通过鉴定靶基因中的突变来解决这个问题，这些突变有助于靶基因在给定的重组宿主中的功能表达。本研究通过三轮随机点突变定向进化和筛选，在酿酒酵母中获得了总酶活提高40倍的菌株。酿酒酵母培养物上清液与野生型相比，如在ABTS [2，2 '-连氮双（3-乙基苯并噻唑啉-6-磺酸）]上测量的（260单位/1/OD 600）。野生型和两个高活性克隆的基因在毕赤酵母中表达，其中总ABTS活性在摇瓶中达到600单位/I/OD 600。突变体对ABTS的比活性高达5.4倍，对愈创木酚的比活性高达2.3倍。
The ability to engineer proteins by directed evolution requires functional expression of the target polypeptide in a recombinant host suitable for construction and screening libraries of enzyme variants. Bacteria and yeast are preferred, but eukaryotic proteins often fail to express in active form in these cells. We have attempted to resolve this problem by identifying mutations in the target gene that facilitate its functional expression in a given recombinant host. Here we examined expression of HRP in Saccharomyces cerevisiae, Through three rounds of directed evolution by random point mutagenesis and screening, we obtained a 40-fold increase in total HRP activity in the S. cerevisiae culture supernatant compared with wild-type, as measured on ABTS [2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)] (260 units/I/OD600). Genes from wild-type and two high-activity clones were expressed in Pichia pastoris, where the total ABTS activity reached 600 units/I/OD600 in shake flasks. The mutants show up to 5.4-fold higher specific activity towards ABTS and 2.3-fold higher specific activity towards guaiacol.