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首页> 外文期刊>Plasmid: An International Journal Devoted to Extrachromosomal Gene Systems >CONSTRUCTION AND CHARACTERIZATION OF VERSATILE CLONING VECTORS FOR EFFICIENT DELIVERY OF NATIVE FOREIGN PROTEINS TO THE PERIPLASM OF ESCHERICHIA COLI
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CONSTRUCTION AND CHARACTERIZATION OF VERSATILE CLONING VECTORS FOR EFFICIENT DELIVERY OF NATIVE FOREIGN PROTEINS TO THE PERIPLASM OF ESCHERICHIA COLI

机译：天然外源蛋白高效传递到大肠杆菌周围的多功能克隆载体的构建和鉴定

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Induction of the wild type cholera toxin operon (ctxAB) from multicopy clones in Escherichia coli inhibited growth and resulted in low yields of cholera toxin (CT). We found that production of wild type CT or its B subunit (CT-B) as a periplasmic protein was toxic for E. coli, but by replacing the native signal sequences of both CT-A and CT-B with the signal sequence from the B subunit of E. coli heat-labile enterotoxin LTIIb, we succeeded for the first time in producing CT holotoxin in high yield in E. coli. Based on these findings, we designed and constructed versatile cloning vectors that use the LTIIb-B signal sequence to direct recombinant native proteins with high efficiency to the periplasm of E. coli. We confirmed the usefulness of these vectors by producing two other secreted recombinant proteins. First, using phoA from E. coli, we demonstrated that alkaline phosphatase activity was 17-fold greater when the LTIIb-B signal sequence was used than when the native leader for alkaline phosphatase was used. Second, using the pspA gene that encodes pneumococcal surface protein A from Streptococcus pneumoniae, we produced a 299-residue amino-terminal fragment of PspA in E. coli in large amounts as a soluble periplasmic protein and showed that it was immunoreactive in Western blots with antibodies against native PspA. The vectors described here will be useful for further studies on structure-function relationships and vaccine development with CT and PspA, and they should be valuable as general tools for delivery of other secretion-competent recombinant proteins to the periplasm in E. coli. (C) 1997 Academic Press. [References: 40]
机译：在大肠杆菌中从多拷贝克隆中诱导野生型霍乱毒素操纵子（ctxAB）会抑制生长，并导致霍乱毒素（CT）的低收率。我们发现，野生型CT或其B亚基（CT-B）作为周质蛋白的生产对大肠杆菌是有毒的，但是通过用来自C1的信号序列代替CT-A和CT-B的天然信号序列。大肠杆菌不耐热肠毒素LTIIb的B亚基，我们首次成功在大肠杆菌中高产生产CT全息毒素。基于这些发现，我们设计并构建了多功能克隆载体，该载体使用LTIIb-B信号序列将重组天然蛋白高效导入到大肠杆菌的周质中。我们通过产生另外两种分泌的重组蛋白证实了这些载体的有用性。首先，使用来自大肠杆菌的phoA，我们证明了使用LTIIb-B信号序列时，碱性磷酸酶活性比使用碱性磷酸酶的天然前导序列高17倍。其次，使用编码肺炎链球菌肺炎球菌表面蛋白A的pspA基因，我们在大肠杆菌中产生了299个残基的PspA氨基末端片段，为可溶性周质蛋白，并显示了它在Western blot中的免疫反应性。天然PspA抗体。此处描述的载体可用于进一步研究结构功能关系以及使用CT和PspA进行疫苗开发，它们应作为将其他具有分泌能力的重组蛋白传递到大肠杆菌周质中的通用工具而有价值。 （C）1997学术出版社。 [参考：40]

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