Simmons L C, Yansura D G
Department of Molecular Biology, Genentech, Inc., South San Francisco, CA 94080, USA.
Nat Biotechnol. 1996 May;14(5):629-34. doi: 10.1038/nbt0596-629.
A method for enhancing the secretion of heterologous proteins in Escherichia coli by optimizing, as opposed to simply maximizing, the translational level of a given protein is described. Random alteration of the translational initiation region (TIR) of the Heat-Stable Enterotoxin II (STII) signal sequence resulted in a library of vectors with varied translational strengths. Subsequent screening of this library using E. coli alkaline phosphatase as a reporter led to the selection of several TIR variants covering a 10-fold range of translational strength. These TIR variants, in combination with several previously generated variants, are shown to dramatically improve the secretion of a number of heterologous proteins. In fact, the heterologous proteins tested required a narrow translational range for optimal high-level secretion into the periplasm. Interestingly, the secretion of two native E. coli proteins was unaffected by TIR strength when tested over an identical range. The dependence of secretion on a narrow translational level demonstrates its critical role in the secretion of heterologous proteins in E. coli.
本文描述了一种通过优化而非简单最大化给定蛋白质的翻译水平来增强大肠杆菌中异源蛋白质分泌的方法。对热稳定肠毒素II（STII）信号序列的翻译起始区域（TIR）进行随机改变，得到了一系列具有不同翻译强度的载体文库。随后以大肠杆菌碱性磷酸酶作为报告基因对该文库进行筛选，从而选出了几种翻译强度范围达10倍的TIR变体。这些TIR变体与之前产生的几种变体相结合，显著提高了多种异源蛋白质的分泌水平。实际上，所测试的异源蛋白质需要在较窄的翻译范围内才能实现向周质的最佳高水平分泌。有趣的是，在相同范围内进行测试时，两种天然大肠杆菌蛋白质的分泌不受TIR强度的影响。分泌对较窄翻译水平的依赖性表明其在大肠杆菌中异源蛋白质分泌过程中起着关键作用。
