High-level accumulation of a recombinant antibody fragment in the periplasm of Escherichia coli requires a triple-mutant (degP prc spr) host strain
High-level accumulation of a recombinant antibody fragment in the periplasm of Escherichia coli requires a triple-mutant (degP prc spr) host strain
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DOI：
10.1002/bit.20014
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发表时间：
2004-03-05
影响因子：
3.8
通讯作者：
Champion, KM
中科院分区：
文献类型：
--
作者：
Chen, C;Snedecor, B;Champion, KM
在生产一种分泌到大肠杆菌周质中的人源化抗体片段过程中，观察到了轻链的蛋白水解降解。为了确定是哪种（些）蛋白酶导致了这种降解，我们比较了在几种周质蛋白酶编码基因发生突变的大肠杆菌菌株中F(ab')(2)抗体片段的表达情况。对在高细胞密度发酵中培养的菌株进行分析表明，degP、prc和spr基因的突变组合对于细胞产生高水平的所需重组抗体片段是必要的。为了消除其他基因突变可能产生的影响，我们在同基因背景下构建了具有蛋白酶突变的大肠杆菌菌株，并在高细胞密度发酵中重复了这些研究。在degP菌株中，广泛的轻链蛋白水解现象仍然存在。然而，在prc和prc spr菌株中，轻链蛋白水解显著减少，并且在prc和prc spr突变菌株中引入degP突变后，水解进一步减少。这些结果表明，在大肠杆菌中表达重组抗体片段期间，周质蛋白酶Prc（Tsp）是轻链蛋白水解降解的主要原因，并且DegP（HtrA）对这种降解也有较小的贡献。结果还表明，spr是prc菌株生长缺陷的抑制因子，prc突变体在整个高细胞密度发酵过程中存活需要它。（C）2004威利期刊公司
During production of a humanized antibody fragment secreted into the periplasm of Escherichia coli, proteolytic degradation of the light chain was observed. In order to determine which protease(s) were responsible for this degradation, we compared expression of the F(ab')(2) antibody fragment in several E. coli strains carrying mutations in genes encoding periplasmic proteases. Analysis of strains cultured in high cell density fermentations showed that the combination of mutations in degP prc spr was necessary for the cells to produce high levels of the desired recombinant antibody fragment. In order to eliminate the possible effects of mutations in other genes, we constructed E. coli strains with protease mutations in isogenic backgrounds and repeated the studies in high cell density fermentations. Extensive light chain proteolysis persisted in degP strains. However, light chain proteolysis was substantially decreased in jorc and jorc spr strains, and was further decreased with the introduction of a degP mutation in prc and prc spr mutant strains. These results show that the periplasmic protease Prc (Tsp) is primarily responsible for proteolytic degradation of the light chain during expression of a recombinant antibody fragment in E. coli, and that DegP (HtrA) makes a minor contribution to this degradation as well. The results also show that spr, a suppressor of growth defects in prc strains, is required for a prc mutant to survive throughout high cell density fermentations. (C) 2004 Wiley Periodicals, Inc.