Jobling M G, Palmer L M, Erbe J L, Holmes R K
Department of Microbiology, University of Colorado Health Sciences Center, Denver 80262, USA.
Plasmid. 1997;38(3):158-73. doi: 10.1006/plas.1997.1309.
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.
在大肠杆菌中，多拷贝克隆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）产量低下。我们发现，作为周质蛋白生产野生型CT或其B亚基（CT-B）对大肠杆菌有毒性，但通过用大肠杆菌不耐热肠毒素LTIIb的B亚基的信号序列替换CT-A和CT-B的天然信号序列，我们首次成功地在大肠杆菌中高产生产了CT全毒素。基于这些发现，我们设计并构建了通用克隆载体，该载体利用LTIIb-B信号序列将重组天然蛋白高效地导向大肠杆菌的周质。我们通过生产另外两种分泌型重组蛋白证实了这些载体的实用性。首先，使用来自大肠杆菌的phoA，我们证明当使用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时高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，我们在大肠杆菌中大量生产了PspA的299个残基的氨基末端片段，作为可溶性周质蛋白，并表明它在Western印迹中与抗天然PspA的抗体具有免疫反应性。本文所述的载体将有助于对CT和PspA的结构-功能关系及疫苗开发进行进一步研究，并且作为将其他具有分泌能力的重组蛋白递送至大肠杆菌周质的通用工具，它们应该具有很高的价值。
