Zhao Hong Liang, Xue Chong, Wang Yang, Yao Xue Qin, Liu Zhi Min
Department of Microorganism Engineering, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai district, Beijing, 100071, People's Republic of China.
Appl Microbiol Biotechnol. 2008 Nov;81(2):235-41. doi: 10.1007/s00253-008-1666-0. Epub 2008 Aug 27.
In this study, we assessed the potential of PMR1-disrupted Pichia pastoris (Pppmr1) expressing human serum albumin and interferon alpha 2b fusion protein (HSA-IFN-alpha 2b) in large-scale fermentation. The high osmotic pressure of standard basal salts medium (BSM) was detrimental to the growth and viability of Pppmr1. HSA-IFN-alpha 2b was secreted into a supernatant with a concentration of up to 112 mg/L after 20 h of induction and then began to decline. In vitro stability tests indicated that the disappearance of HSA-IFN-alpha 2b was ascribed to proteolytic degradation. Decreasing the salt concentration of BSM medium to one quarter of the original formula improved the growth and viability of Pppmr1. As a result of reduced cell lysis and protease release, HSA-IFN-alpha 2b was stable in the supernatant, which enabled a longer production phase (30 h) and a higher expression level (215 mg/L). Lowering the culture temperature to 20 degrees C increased the cell viability during carbon source transition and alleviated the oxygen and methanol limitation, which extended the production phase to 40 h and increased the expression level to 680 mg/L. The addition of 2% Soytone prolonged the production phase to 60 h and increased the expression level to 1,260 mg/L, which was more than tenfold higher than that of Pppmr1 cultured under the conditions recommended by Invitrogen.
在本研究中，我们评估了表达人血清白蛋白和干扰素α 2b融合蛋白（HSA-IFN-α 2b）的PMR1基因缺失型毕赤酵母（Pppmr1）在大规模发酵中的潜力。标准基础盐培养基（BSM）的高渗透压对Pppmr1的生长和活力不利。诱导20小时后，HSA-IFN-α 2b分泌到上清液中，浓度高达112 mg/L，随后开始下降。体外稳定性测试表明，HSA-IFN-α 2b的消失归因于蛋白水解降解。将BSM培养基的盐浓度降低至原配方的四分之一，可改善Pppmr1的生长和活力。由于细胞裂解和蛋白酶释放减少，HSA-IFN-α 2b在上清液中稳定，这使得生产阶段延长（30小时）且表达水平更高（215 mg/L）。将培养温度降至20℃可提高碳源转换期间的细胞活力，并缓解氧气和甲醇限制，从而将生产阶段延长至40小时，并将表达水平提高至680 mg/L。添加2%大豆蛋白胨可将生产阶段延长至60小时，并将表达水平提高至1260 mg/L，这比在Invitrogen推荐条件下培养的Pppmr1高出十倍以上。
