Kaderbhai Mustak A, Davey Hazel M, Kaderbhai Naheed N
Institute of Biological Sciences, Cledwyn Building, University of Wales, Aberystwyth, Ceredigion, SY23 3DD, UK.
Protein Sci. 2004 Sep;13(9):2458-69. doi: 10.1110/ps.04697304.
A directed evolutionary approach is described that searches short, random peptide sequences for appendage at the secretory signal peptide-mature protein junction to seek ideal algorithms for both efficient and hyper export of recombinant proteins to the periplasm of Escherichia coli. The strategy employs simple, visual detection of positive clones using a PINK expression system that faithfully reports on export status of a mammalian hemoprotein in E. coli. With-in "sequence spaces" ranging from 1 to 13 residues, a significant but highly variable secretory fitness was scored such that the rate of secretion reciprocally correlated with the membrane-associated precursor pool of the evolved exportable hemoproteins. Three clusters of hyper, median, and hypo exporters were isolated. These had corresponding net charges of -1, 0, and +1 within the evolved sequence space, which in turn clearly correlated with the prevailing magnitude and polarity of the membrane energization states. The findings suggest that both the nature of the charged residue and the proximal sequence in the early mature region are the crucial determinants of the protonophore-dependent electrophoretic discharge of the precursor across the inner membrane of E. coli. We conclude that the directed evolutionary approach will find ready application in engineering recombinant proteins for their efficient secretion via the sec export pathway in E. coli.
本文描述了一种定向进化方法，该方法在分泌信号肽 - 成熟蛋白连接处搜索短的随机肽序列以寻找附加物，从而寻求将重组蛋白高效且超量输出到大肠杆菌周质的理想算法。该策略使用一种PINK表达系统，通过简单的视觉检测阳性克隆，该系统能如实报告哺乳动物血红蛋白在大肠杆菌中的输出状态。在1至13个残基的“序列空间”内，对显著但高度可变的分泌适应性进行了评分，使得分泌速率与进化后的可输出血红蛋白的膜相关前体池呈反比关系。分离出了三组超量、中等和少量输出者。在进化后的序列空间内，它们相应的净电荷分别为 -1、0和 +1，这又与膜通电状态的主要大小和极性明显相关。研究结果表明，带电残基的性质以及早期成熟区域的近端序列是前体通过大肠杆菌内膜进行质子载体依赖性电泳释放的关键决定因素。我们得出结论，定向进化方法将在通过大肠杆菌的sec输出途径对重组蛋白进行高效分泌工程中得到广泛应用。
