Studies on gamma-Glutamyltranspeptidase of Escherichia coli

1.测定了大肠杆菌K-12 γ-谷氨酰转肽酶（GGT）基因的核苷酸序列.该序列包含编码信号肽（25个氨基酸残基）的单个开放阅读框以及按该顺序的大亚基（365个）和小亚基（190个）。这一结果表明E.大肠杆菌γ-谷氨酰转肽酶是后处理的，如哺乳动物GGT的情况。将其氨基酸序列与哺乳动物的GGT进行比较，发现E. coliGGT与哺乳动物GGT有30%的同源性，大部分蛋白是保守的，通过氨基酸的保守替换.采用简单的两步法从大肠杆菌中大量纯化GGT。coliSH 643，含有ggt克隆的质粒pSH101.3。以GGT为原料合成了S-苄基谷胱甘肽甲酯、γ-谷氨酰-L-酪氨酸甲酯和γ-谷氨酰-L-组氨酸，产率分别为31.2、35.7和41.2 g/L.通过以下两步法将ggt结构基因克隆到高效表达载体pKK 223 -3中。首先，将编码该大亚基的EcoRV片段插入载体质粒中。然后尝试将编码小亚基的ggt的HpaI-Pst I片段插入到具有EcoRV片段的载体中。现在我们已经成功地迈出了第一步.采用定点突变的方法将小亚基中的Ser 75残基替换为Ala或Cys。突变体细胞表现出与野生型几乎相同的活性。突变酶的性质正在研究中。

1. The nucleotide sequence of ggt, the gene that codes for gamma -glutamyltrans- peptidase(GGT) of Escerichia coli K-12, was determined. The sequence contains a single open reading frame encoding the signal peptide(25 amino acid residues) and large(365) and small(190) subunits, in that order. This result suggests that E. coli gamma-glutamyltranspeptidase is processed posttranslationally, as in the case of mammalian GGTs. The amino acids sequence was compared with those of mammalian GGTs and we found that E. coli GGT has 30% homology with mammalian ones and most part of the protein is conserved through conservative substitutions of amino acid.2. By a simple two-step method, a large amount of GGT was purified from E. coli SH643 harboring ggt-cloned plassid pSH101.3. S-Benzyl glutathione methyl ester, gamma-glutamyl-L-tyrosine methylester and gamma - glutamyl-L-histidine were synthesized by GGT in the yield of 31.2, 35.7 and 41.2 g/L, respectively.4. Cloning of the structure gene of ggt into the high-expression vector pKK223-3 was carried out by the following two-step method. First, EcoRV fragment which encodes the large subunit was inserted into the vector plasmid. And then the HpaI-PstI fragment of ggt which encodes small subunit was tried to insert into the vector having the EcoRV fragment. Now we are successful in the first step.5. Ser75 residue in the small subunit was replaced with Ala or Cys by the method of site directed mutation. The mutant cells exhibited almost same activity with the wild type. The properties of mutant enzymes are under investigation now.