Munro A W, Ritchie G Y, Lamb A J, Douglas R M, Booth I R
Department of Molecular and Cell Biology, Marischal College, University of Aberdeen, UK.
Mol Microbiol. 1991 Mar;5(3):607-16. doi: 10.1111/j.1365-2958.1991.tb00731.x.
The kefC gene of Escherichia coli encodes a potassium-efflux system that is regulated by glutathione metabolites. The close proximity of the E. coli kefC gene to the folA gene, encoding dihydrofolate reductase, has been utilized to clone the structural gene for the system from a Clarke-Carbon plasmid. The cloned gene has been refined to a region of DNA approximately 2.1 kb in length using exonuclease III-generated deletions and random MudII1734 (lacZ) insertions. The direction of transcription has been deduced from the orientation of the Mu insertions in the cloned DNA. A hybrid protein consisting of approximately two thirds of the KefC protein fused to beta-galactosidase has been shown to be membrane-located. The DNA sequence of the gene has been determined and an open reading frame of 1.86 kb has been located which could encode a protein of 620 amino acids (79010 Da). Using the T7 expression system a membrane protein, of apparent molecular mass 55-60 kDa, has been shown to be encoded by the kefC gene. The predicted protein sequence shows a highly hydrophobic amino-terminus and a strongly hydrophilic carboxy-terminus. Comparison of the amino acid sequence of the kefC gene product with those of two glutathione-utilizing enzymes, glyoxalase and dehalogenase, has revealed some similarities.
大肠杆菌的kefC基因编码一种受谷胱甘肽代谢物调控的钾离子外流系统。利用大肠杆菌kefC基因与编码二氢叶酸还原酶的folA基因的紧密相邻关系，从克拉克-卡尔文质粒中克隆了该系统的结构基因。通过核酸外切酶III产生的缺失和随机的MudII1734（lacZ）插入，将克隆基因精细定位到一段长度约为2.1 kb的DNA区域。转录方向已根据克隆DNA中Mu插入的方向推导出来。一种由约三分之二的KefC蛋白与β-半乳糖苷酶融合而成的杂合蛋白已被证明定位于细胞膜。已确定该基因的DNA序列，并定位到一个1.86 kb的开放阅读框，它可能编码一个620个氨基酸（79010 Da）的蛋白质。使用T7表达系统已证明kefC基因编码一种表观分子量为55 - 60 kDa的膜蛋白。预测的蛋白质序列显示出高度疏水的氨基末端和强亲水的羧基末端。将kefC基因产物的氨基酸序列与两种利用谷胱甘肽的酶——乙二醛酶和脱卤酶的氨基酸序列进行比较，发现了一些相似之处。
