Browning Douglas F, Richards Kirsty L, Peswani Amber R, Roobol Jo, Busby Stephen J W, Robinson Colin
Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, UK.
School of Biosciences, University of Kent, Canterbury, UK.
Biotechnol Bioeng. 2017 Dec;114(12):2828-2836. doi: 10.1002/bit.26434. Epub 2017 Oct 6.
Numerous high-value proteins are secreted into the Escherichia coli periplasm by the General Secretory (Sec) pathway, but Sec-based production chassis cannot handle many potential target proteins. The Tat pathway offers a promising alternative because it transports fully folded proteins; however, yields have been too low for commercial use. To facilitate Tat export, we have engineered the TatExpress series of super-secreting strains by introducing the strong inducible bacterial promoter, ptac, upstream of the chromosomal tatABCD operon, to drive its expression in E. coli strains commonly used by industry (e.g., W3110 and BL21). This modification significantly improves the Tat-dependent secretion of human growth hormone (hGH) into the bacterial periplasm, to the extent that secreted hGH is the dominant periplasmic protein after only 1 hr induction. TatExpress strains accumulate in excess of 30 mg L periplasmic recombinant hGH, even in shake flask cultures. A second target protein, an scFv, is also shown to be exported at much higher rates in TatExpress strains.
许多高价值蛋白质通过通用分泌（Sec）途径分泌到大肠杆菌周质中，但基于Sec的生产底盘无法处理许多潜在的目标蛋白质。Tat途径提供了一种有前景的替代方案，因为它运输完全折叠的蛋白质；然而，产量一直过低，无法用于商业用途。为了促进Tat输出，我们通过在染色体tatABCD操纵子上游引入强诱导型细菌启动子ptac，构建了TatExpress系列超分泌菌株，以驱动其在工业常用的大肠杆菌菌株（如W3110和BL21）中表达。这种修饰显著提高了人生长激素（hGH）向细菌周质的Tat依赖性分泌，以至于在仅诱导1小时后，分泌的hGH就成为主要的周质蛋白。即使在摇瓶培养中，TatExpress菌株积累的周质重组hGH也超过30 mg/L。另一种目标蛋白scFv在TatExpress菌株中的输出率也显著更高。
