How to find soluble proteins: a comprehensive analysis of alpha/beta hydrolases for recombinant expression in E. coli.
How to find soluble proteins: a comprehensive analysis of alpha/beta hydrolases for recombinant expression in E. coli.
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DOI：
10.1186/1471-2164-6-49
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发表时间：
2005-04-02
期刊：
影响因子：
4.4
通讯作者：
Pleiss, Jurgen
中科院分区：
文献类型：
--
作者：
Koschorreck, Markus;Fischer, Markus;Barth, Sandra;Pleiss, Jurgen
在表达克隆文库的筛选中，活性蛋白在E.大肠杆菌中的包涵体的形成是限制性的。在这些情况下，需要在大肠杆菌中富集具有可溶性基因产物的编码序列的基因文库。从而提高筛选效率。以前，威尔金森和哈里森表明，可以从氨基酸组成预测溶解度（Biotechnology 1991，9（5）：443-448）。我们已经将该分析应用于α/β水解酶折叠家族的成员，以预测它们在E.杆菌α/β水解酶是一个高度多样化的家族，具有超过1800种蛋白质，这些蛋白质被分为同源家族和超家族。在E.大肠杆菌中的水解酶取决于水解酶的大小、宿主生物的系统发育起源、水解酶所属的同源家族和超家族。一般来说，小的水解酶比大的水解酶更易溶，而真核水解酶在大肠杆菌中的溶解度较低。coli比原核生物的要多。然而，结合系统发育起源和大小导致更复杂的结论。来自原核生物、真菌和后生动物来源的水解酶如果分别具有小、中和大尺寸，则预测它们是最可溶的。我们观察到不同同源家族和不同类群的水解酶之间的预测溶解度的变化很大。通过使用含有更多可溶性基因产物的文库，对所有α/β水解酶序列的综合分析允许更有效地筛选新的可溶性α/β水解酶。筛选在大肠杆菌中难以表达的水解酶家族。大肠杆菌中的蛋白质应该首先在来自系统发育组的生物体的编码序列中进行，该系统发育组具有该家族蛋白质的最高平均预测溶解度。这里开发的工具可用于使用数据库中公布的蛋白质序列来识别用于表达的有吸引力的靶基因。该分析还指导简并的家族特异性引物的设计，以扩增来自同源家族或超家族的新成员，其中可溶性α/β水解酶的可能性高。
In screening of libraries derived by expression cloning, expression of active proteins in E. coli can be limited by formation of inclusion bodies. In these cases it would be desirable to enrich gene libraries for coding sequences with soluble gene products in E. coli and thus to improve the efficiency of screening. Previously Wilkinson and Harrison showed that solubility can be predicted from amino acid composition (Biotechnology 1991, 9(5):443–448). We have applied this analysis to members of the alpha/beta hydrolase fold family to predict their solubility in E. coli. alpha/beta hydrolases are a highly diverse family with more than 1800 proteins which have been grouped into homologous families and superfamilies. The predicted solubility in E. coli depends on hydrolase size, phylogenetic origin of the host organism, the homologous family and the superfamily, to which the hydrolase belongs. In general small hydrolases are predicted to be more soluble than large hydrolases, and eukaryotic hydrolases are predicted to be less soluble in E. coli than prokaryotic ones. However, combining phylogenetic origin and size leads to more complex conclusions. Hydrolases from prokaryotic, fungal and metazoan origin are predicted to be most soluble if they are of small, medium and large size, respectively. We observed large variations of predicted solubility between hydrolases from different homologous families and from different taxa. A comprehensive analysis of all alpha/beta hydrolase sequences allows more efficient screenings for new soluble alpha/beta hydrolases by the use of libraries which contain more soluble gene products. Screening of hydrolases from families whose members are hard to express as soluble proteins in E. coli should first be done in coding sequences of organisms from phylogenetic groups with the highest average of predicted solubility for proteins of this family. The tools developed here can be used to identify attractive target genes for expression using protein sequences published in databases. This analysis also directs the design of degenerate, family- specific primers to amplify new members from homologous families or superfamilies with a high probability of soluble alpha/beta hydrolases.