Rapid screening for improved solubility of small human proteins produced as fusion proteins in Escherichia coli
Rapid screening for improved solubility of small human proteins produced as fusion proteins in Escherichia coli
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DOI：
10.1110/ps.22102
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发表时间：
2002-02-01
期刊：
影响因子：
8
通讯作者：
Härd, T
中科院分区：
文献类型：
--
作者：
Hammarström, M;Hellgren, N;Härd, T
结构基因组学和相关项目的一个先决条件是标准化基因过度表达和蛋白质溶解性筛选的过程，以实现自动化以获得更高的产量。我们已经测试了一种快速亚克隆大量人类基因的方法，并对这些基因在大肠杆菌中的表达和蛋白质溶解性进行了筛选。这种可以部分自动化的方法被用来比较六种不同的N端融合蛋白和N端6*His标签的效果。作为一个现实的测试集，我们选择了32个结构和大小未知的、适合于核磁共振研究的潜在有趣的人类蛋白质。通过重组亚克隆将基因从cDNA3转移到表达载体上。在32个基因中，27个基因的亚克隆得率为100%，可获得大小正确的PCR片段。其中，26个基因(96%)可以在可检测到的水平上过度表达，23个基因(85%)在至少一个融合标签的可溶性部分中被检测到。我们发现融合蛋白或TAG对表达和溶解性的影响有很大的差异。简而言之，七个融合中的四个表现非常好，而且比6*His标签要好得多，但个体差异促使在表达和溶解性筛选中纳入几个融合。我们还得出结论，我们的方法和表达载体可以用于结构研究的基因筛选，并且应该有可能在大肠杆菌中获得大部分核磁共振大小的非膜人类蛋白作为可溶性融合蛋白。
A prerequisite for structural genomics and related projects is to standardize the process of gene overexpression and protein solubility screening to enable automation for higher throughput. We have tested a methodology to rapidly subelone a large number of human genes and screen these for expression and protein solubility in Escherichia coli. The methodology, which can be partly automated, was used to compare the effect of six different N-terminal fusion proteins and an N-terminal 6*His tag. As a realistic test set we selected 32 potentially interesting human proteins with unknown structures and sizes suitable for NMR studies. The genes were transferred from cDNA to expression vectors using subcloning by recombination. The subcloning yield was 100% for 27 (of 32) genes for which a PCR fragment of correct size could be obtained. Of these, 26 genes (96%) could be overexpressed at detectable levels and 23 (85%) are detected in the soluble fraction with at least one fusion tag. We find large differences in the effects of fusion protein or tag on expression and solubility. In short, four of seven fusions perform very well, and much better than the 6*His tag, but individual differences motivate the inclusion of several fusions in expression and solubility screening. We also conclude that our methodology and expression vectors can be used for screening of genes for structural studies, and that it should be possible to obtain a large fraction of all NMR-sized and nonmembrane human proteins as soluble fusion proteins in E. coli.