State Key Laboratory of Food Science and Technology, The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, Jiangsu, China.
Enzyme Microb Technol. 2013 Dec 10;53(6-7):438-43. doi: 10.1016/j.enzmictec.2013.09.009. Epub 2013 Sep 30.
Pichia pastoris has been successfully used in the production of many secreted and intracellular recombinant proteins, but there is still a large room of improvement for this expression system. Two factors drastically influence the lipase r27RCL production from Rhizopus chinensis CCTCC M201021, which are gene dosage and protein folding in the endoplasmic reticulum (ER). Regarding the effect of gene dosage, the enzyme activity for recombinant strain with three copies lipase gene was 1.95-fold higher than that for recombinant strain with only one copy lipase gene. In addition, the lipase production was further improved by co-expression with chaperone PDI involved in the disulfide bond formation in the ER. Overall, the maximum enzyme activity reached 355U/mL by the recombinant strain with one copy chaperone gene PDI plus five copies lipase gene proRCL in shaking flasks, which was 2.74-fold higher than that for the control strain with only one copy lipase gene. Overall, co-expression with PDI vastly increased the capacity for processing proteins of ER in P. pastoris.
毕赤酵母已成功用于许多分泌型和胞内重组蛋白的生产，但该表达系统仍有很大的改进空间。有两个因素极大地影响了从里氏木霉 CCTCC M201021 生产脂肪酶 r27RCL，它们是基因剂量和内质网（ER）中的蛋白质折叠。关于基因剂量的影响，带有三个拷贝脂肪酶基因的重组菌的酶活比仅带有一个拷贝脂肪酶基因的重组菌高 1.95 倍。此外，通过与涉及 ER 中二硫键形成的伴侣蛋白 PDI 共表达进一步提高了脂肪酶的产量。总的来说，在摇瓶中，带有一个拷贝伴侣蛋白基因 PDI 和五个拷贝脂肪酶基因 proRCL 的重组菌的最大酶活达到 355U/mL，比仅带有一个拷贝脂肪酶基因的对照菌高 2.74 倍。总的来说，与 PDI 共表达极大地提高了毕赤酵母 ER 中蛋白质加工的能力。
