Moua Pachai S, Gonzalez Alfonso, Oshiro Kristin T, Tam Vivian, Li Zhiguo Harry, Chang Jennifer, Leung Wilson, Yon Amy, Thor Der, Venkatram Sri, Franz Andreas H, Risser Douglas D, Lin-Cereghino Joan, Lin-Cereghino Geoff P
Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, United States.
Department of Chemistry, University of the Pacific, Stockton, CA 95211, United States.
Protein Expr Purif. 2016 Aug;124:1-9. doi: 10.1016/j.pep.2016.04.005. Epub 2016 Apr 11.
The Escherichia coli maltose binding protein (MBP) is an N-terminal fusion partner that was shown to enhance the secretion of some heterologous proteins from the yeast Pichia pastoris, a popular host for recombinant protein expression. The amount of increase in secretion was dependent on the identity of the cargo protein, and the fusions were proteolyzed prior to secretion, limiting its use as a purification tag. In order to overcome these obstacles, we used the MBP as C-terminal partner for several cargo peptides. While the Cargo-MBP proteins were no longer proteolyzed in between these two moieties when the MBP was in this relative position, the secretion efficiency of several fusions was lower than when MBP was located at the opposite end of the cargo protein (MBP-Cargo). Furthermore, fluorescence analysis suggested that the MBP-EGFP and EGFP-MBP proteins followed different routes within the cell. The effect of several Pichia pastoris beta-galactosidase supersecretion (bgs) strains, mutants showing enhanced secretion of select reporters, was also investigated on both MBP-EGFP and EGFP-MBP. While the secretion efficiency, proteolysis and localization of the MBP-EGFP was influenced by the modified function of Bgs13, EGFP-MBP behavior was not affected in the bgs strain. Taken together, these results indicate that the location of the MBP in a fusion affects the pathway and trans-acting factors regulating secretion in P. pastoris.
大肠杆菌麦芽糖结合蛋白（MBP）是一种N端融合伴侣，已证明它能增强酵母毕赤酵母（一种常用的重组蛋白表达宿主）中某些异源蛋白的分泌。分泌增加的量取决于负载蛋白的特性，并且融合蛋白在分泌前会被蛋白酶水解，这限制了它作为纯化标签的用途。为了克服这些障碍，我们将MBP用作几种负载肽的C端伴侣。当MBP处于这个相对位置时，负载 - MBP蛋白在这两个部分之间不再被蛋白酶水解，然而几种融合蛋白的分泌效率低于MBP位于负载蛋白另一端（MBP - 负载）时的情况。此外，荧光分析表明，MBP - EGFP和EGFP - MBP蛋白在细胞内遵循不同的途径。还研究了几种毕赤酵母β - 半乳糖苷酶超分泌（bgs）菌株（显示某些报告基因分泌增强的突变体）对MBP - EGFP和EGFP - MBP的影响。虽然Bgs13的修饰功能影响了MBP - EGFP的分泌效率、蛋白水解和定位，但EGFP - MBP在bgs菌株中的行为不受影响。综上所述，这些结果表明MBP在融合蛋白中的位置会影响毕赤酵母中调节分泌的途径和反式作用因子。
