Veeravalli Karthik, Laird Michael W, Fedesco Mark, Zhang Yu, Yu X Christopher
Dept. of Late Stage Cell Culture, Genentech Inc., South San Francisco, CA, 94080.
Biotechnol Prog. 2015 Jan-Feb;31(1):204-11. doi: 10.1002/btpr.1999. Epub 2014 Oct 21.
Incorporation of norleucine in place of methionine residues during recombinant protein production in Escherichia coli is well known. Continuous feeding of methionine is commonly used in E. coli recombinant protein production processes to prevent norleucine incorporation. Although this strategy is effective in preventing norleucine incorporation, there are several disadvantages associated with continuous feeding. Continuous feeding increases the operational complexity and the overall cost of the fermentation process. In addition, the continuous feed leads to undesirable dilution of the fermentation medium possibly resulting in lower cell densities and recombinant protein yields. In this work, the genomes of three E. coli hosts were engineered by introducing chromosomal mutations that result in methionine overproduction in the cell. The recombinant protein purified from the fermentations using the methionine overproducing hosts had no norleucine incorporation. Furthermore, these studies demonstrated that the fermentations using one of the methionine overproducing hosts exhibited comparable fermentation performance as the control host in three different recombinant protein production processes.
在大肠杆菌中进行重组蛋白生产时，用正亮氨酸取代甲硫氨酸残基是众所周知的。在大肠杆菌重组蛋白生产过程中，通常采用连续添加甲硫氨酸的方法来防止正亮氨酸的掺入。尽管这种策略在防止正亮氨酸掺入方面是有效的，但连续添加也存在几个缺点。连续添加会增加操作复杂性和发酵过程的总体成本。此外，连续进料会导致发酵培养基的不必要稀释，可能导致细胞密度和重组蛋白产量降低。在这项工作中，通过引入导致细胞中甲硫氨酸过量产生的染色体突变，对三种大肠杆菌宿主的基因组进行了改造。使用甲硫氨酸过量产生宿主进行发酵纯化得到的重组蛋白没有正亮氨酸掺入。此外，这些研究表明，在三种不同的重组蛋白生产过程中，使用其中一种甲硫氨酸过量产生宿主进行的发酵表现出与对照宿主相当的发酵性能。
