A system for concomitant overexpression of four periplasmic folding catalysts to improve secretory protein production in Escherichia coli
A system for concomitant overexpression of four periplasmic folding catalysts to improve secretory protein production in Escherichia coli
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DOI：
10.1093/protein/gzl018
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发表时间：
2006-08-01
影响因子：
2.4
通讯作者：
Skerra, Arne
中科院分区：
文献类型：
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作者：
Schlapschy, Martin;Grimm, Sebastian;Skerra, Arne
虽然大肠杆菌广泛用于制备蛋白表达，但在重组基因产物的折叠和蛋白质聚集方面经常遇到问题。除了细胞质表达外，分泌到细菌周质中也是如此，细菌周质是生产带有结构二硫键的蛋白质的首选方法。在这里，我们报道了辅助载体pTUM4的构建，它影响了四种已建立的周质伴侣和折叠催化剂的过度表达：催化二硫键形成和异构化的硫醇-二硫化物氧化还原酶DsbA和DsbC，以及具有伴侣活性的肽基-脯氨基顺式/反式异构酶FkpA和SurA。PTUM4携带P15A复制起始点和氯霉素抗性基因，因此，它与许多使用ColEI起始点和氨苄西林抗性的传统表达载体兼容。利用人血浆视黄醇结合蛋白和树突状细胞膜受体DC-SIGN的胞外碳水化合物识别结构域，说明了其对可溶性重组蛋白产量和二硫键均一性的积极作用。因此，pTUM4代表了一种新的辅助载体，它补充了现有的胞质伴侣共表达载体，并有望用于抑制折叠效率的各种重组蛋白的功能分泌。
Although Escherichia coli is in wide use for preparative protein expression, problems with the folding of the recombinant gene product and protein aggregation are frequently encountered. Apart from cytoplasmic expression, this is also true for secretion into the bacterial periplasm, the method of choice for the production of proteins that carry structural disulfide bonds. Here we report the construction of the helper plasmid pTUM4, which effects overexpression of four established periplasmic chaperones and folding catalysts: the thiol-disulfide oxidoreductases DsbA and DsbC that catalyze the formation and isomerization of disulfide bridges and the peptidyl-prolyl cis/trans-isomerases with chaperone activity, FkpA and SurA. pTUM4 carries a p15a origin of replication and a chloramphenicol resistance gene and, thus, it is compatible with many conventional expression vectors that use the ColEI origin and an ampicillin resistance. Its positive effects on the yield of soluble recombinant protein and the homogeneity of disulfide pattern are illustrated here using the human plasma retinol-binding protein as well as the extracellular carbohydrate recognition domain of the dendritic cell membrane receptor DC-SIGN. Hence, pTUM4 represents a novel helper vector which complements existing cytosolic chaperone coexpression plasmids and should be useful for the functional secretion of various recombinant proteins with hampered folding efficiency.