Bedouelle H, Duplay P
Unité de Biochimie des Régulations Cellulaires (Centre National de la Recherche Scientifique Unité Associée 1129), Institut Pasteur, Paris, France.
Eur J Biochem. 1988 Feb 1;171(3):541-9. doi: 10.1111/j.1432-1033.1988.tb13823.x.
Two enzymes, the secreted Staphylococcus aureus nuclease A and the Klenow fragment of the cytoplasmic Escherichia coli DNA polymerase I, were fused, at the genetic level, to MalE, the periplasmic maltose-binding protein of E. coli, or to a signal-sequence mutant. The hybrid proteins were synthesized in large amounts by E. coli under control of promoter malEp. The synthesis was repressed with glucose and could be totally switched off in a malT mutant strain. The hybrid between MalE and the nuclease was exported into the periplasmic space. Several criteria demonstrated that a fraction of the hybrid chains with the Klenow polymerase was exported to the periplasm in a signal-sequence-specific manner and ruled out the possibility of a membrane leakage. The hybrid with the Klenow polymerase was not exported and remained in the cytoplasm when carrying a tight signal-sequence mutation in its MalE portion. The hybrid proteins were purified in one step by affinity chromatography on cross-linked amylose. Most of the hybrid chains in the periplasm but only a fraction of those in the other cell compartments had their MalE portion correctly folded. The nuclease and the Klenow polymerase had their full specific activities in the purified hybrids. The potential of MalE as a vector for the production, export and purification of desirable proteins in E. coli is discussed.
在基因水平上，将两种酶——分泌型金黄色葡萄球菌核酸酶A和细胞质大肠杆菌DNA聚合酶I的Klenow片段，与大肠杆菌周质麦芽糖结合蛋白MalE或信号序列突变体融合。杂交蛋白在启动子malEp的控制下由大肠杆菌大量合成。合成过程受到葡萄糖的抑制，并且在malT突变株中可完全关闭。MalE与核酸酶之间的杂交体被输出到周质空间。多项标准表明，一部分带有Klenow聚合酶的杂交链以信号序列特异性方式被输出到周质，排除了膜渗漏的可能性。当MalE部分携带紧密信号序列突变时，与Klenow聚合酶的杂交体未被输出并保留在细胞质中。杂交蛋白通过交联直链淀粉亲和层析一步纯化。周质中的大多数杂交链，但其他细胞区室中的只有一部分杂交链，其MalE部分正确折叠。核酸酶和Klenow聚合酶在纯化的杂交体中具有完整的比活性。讨论了MalE作为在大肠杆菌中生产、输出和纯化所需蛋白质的载体的潜力。
