Angele C, Oster T, Visvikis A, Michels J M, Wellman M, Siest G
Centre du Médicament, URA CNRS No. 597, Nancy, France.
Clin Chem. 1991 May;37(5):662-6.
To prepare a reference material for gamma-glutamyltransferase (GGT; EC 2.3.2.2) measurements in clinical chemistry, we constructed different vectors containing either the rat kidney or the human hepatoma Hep G2 GGT cDNA downstream from an inducible promoter for expression in Escherichia coli and Saccharomyces cerevisiae. Transformed bacterial and yeast cells were tested for GGT production by use of Western blot analysis and enzymatic activity measurements. Both rat renal and Hep G2 GGT cDNAs were expressed in E. coli, producing active and nonglycosylated enzymes localized in the periplasmic space. Recombinant Hep G2 GGT was synthesized as a single-chain protein, unlike rat renal GGT, which presented two polypeptides of 62 and 30 kDa, identified as the precursor and a GGT heavy-subunit-like peptide, respectively. Rat renal GGT was produced in S. cerevisiae as two polypeptides, 55 and 30 kDa, detected by antisera against rat renal GGT. These results suggest maturation mechanisms such as glycosylation and cleavage steps, enhancing the interest of S. cerevisiae as a useful expression system for producing active mammalian proteins as reference materials.
为制备临床化学中γ-谷氨酰转移酶（GGT；EC 2.3.2.2）测量的参考物质，我们构建了不同的载体，这些载体含有大鼠肾脏或人肝癌Hep G2 GGT cDNA，位于可诱导启动子下游，用于在大肠杆菌和酿酒酵母中表达。通过蛋白质免疫印迹分析和酶活性测量对转化的细菌和酵母细胞进行GGT产生的测试。大鼠肾脏和Hep G2 GGT cDNA均在大肠杆菌中表达，产生位于周质空间的有活性的非糖基化酶。重组Hep G2 GGT作为单链蛋白合成，与大鼠肾脏GGT不同，大鼠肾脏GGT呈现出62 kDa和30 kDa的两条多肽，分别鉴定为前体和GGT重链亚基样肽。大鼠肾脏GGT在酿酒酵母中产生两条多肽，55 kDa和30 kDa，可被抗大鼠肾脏GGT抗血清检测到。这些结果提示了诸如糖基化和切割步骤等成熟机制，增强了酿酒酵母作为生产活性哺乳动物蛋白作为参考物质的有用表达系统的吸引力。
