THE CLONING AND DNA-SEQUENCE OF THE GENE FOR THE GLUTATHIONE-REGULATED POTASSIUM-EFFLUX SYSTEM KEFC OF ESCHERICHIA-COLI
THE CLONING AND DNA-SEQUENCE OF THE GENE FOR THE GLUTATHIONE-REGULATED POTASSIUM-EFFLUX SYSTEM KEFC OF ESCHERICHIA-COLI
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DOI：
10.1111/j.1365-2958.1991.tb00731.x
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发表时间：
1991-03-01
影响因子：
3.6
通讯作者：
BOOTH, IR
中科院分区：
文献类型：
--
作者：
MUNRO, AW;RITCHIE, GY;BOOTH, IR
大肠杆菌的 kefC 基因编码受谷胱甘肽代谢物调节的钾流出系统。大肠杆菌 kefC 基因与编码二氢叶酸还原酶的 folA 基因非常接近，已被用来从 Clarke-Carbon 质粒中克隆该系统的结构基因。使用核酸外切酶 III 生成的缺失和随机 MudII1734 (IacZ) 插入，将克隆的基因精炼成长度约为 2.1 kb 的 DNA 区域。转录方向是根据克隆 DNA 中 Mu 插入的方向推断出来的。由约三分之二的 kefC 蛋白与 β-半乳糖苷酶融合组成的杂合蛋白已被证明位于膜上。该基因的DNA序列已被确定，并找到一个1.86 kb的开放阅读框，可编码620个氨基酸（79010Da）的蛋白质。使用 T7 表达系统，表观分子量为 55-60kDa 的膜蛋白已被证明是由 kefC 基因编码的。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谷胱甘肽利用酶（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。
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 (IacZ) 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 (79010Da). Using the T7 expression system a membrane protein, of apparent molecular mass 55-60kDa, 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.