Xu Yali, Yasin Amrita, Tang Raymond, Scharer Jeno M, Moo-Young Murray, Chou C Perry
Department of Chemical Engineering, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada, N2L 3G1.
Appl Microbiol Biotechnol. 2008 Nov;81(1):79-87. doi: 10.1007/s00253-008-1644-6. Epub 2008 Aug 29.
Functional expression of lipase B from Pseudozyma antarctica (PalB) in the cytoplasm of Escherichia coli BL21(DE3) and its mutant derivative Origami B(DE3) was explored. Coexpression of DsbA was found to be effective in enhancing PalB expression. The improvement was particularly pronounced with Origami B(DE3) as a host, suggesting that both folding and disulfide bond formation may be major factors limiting PalB expression. Fusion tag technique was also explored by constructing several PalB fusions for the evaluation of their expression performance. While the solubility was enhanced for most PalB fusions, only the DsbA tag was effective in boosting PalB activity, possibly by both enhanced solubility and correct disulfide bond formation. Our results suggest that PalB activity is closely associated with correct disulfide bond formation, and increased solubilization by PalB fusions does not necessarily result in activity enhancement.
探索了南极假丝酵母脂肪酶B（PalB）在大肠杆菌BL21(DE3)及其突变衍生物Origami B(DE3)细胞质中的功能表达。发现共表达DsbA可有效增强PalB的表达。以Origami B(DE3)作为宿主时，这种改善尤为明显，这表明折叠和二硫键形成可能都是限制PalB表达的主要因素。还通过构建几种PalB融合蛋白来探索融合标签技术，以评估它们的表达性能。虽然大多数PalB融合蛋白的溶解度有所提高，但只有DsbA标签能有效提高PalB活性，这可能是通过增强溶解度和形成正确的二硫键实现的。我们的结果表明，PalB活性与正确的二硫键形成密切相关，并且PalB融合蛋白增加的溶解性不一定会导致活性增强。
