Molecular cloning of Escherichia coli K-12 ggt and rapid isolation of gamma-glutamyltranspeptidase.
Based on the results of mapping of ggt, eight strains were selected from a gene library of E. coli. One of the strains harboring pLC9-12 was found to show 14 times higher gamma-glutamyltranspeptidase activity per cell than the wild type strain. The ggt was subcloned to the BamHI site of pUC18 and the recombinant plasmid pSH101 was obtained. Ggt- phenotype of gamma-glutamyltranspeptidase-deficient mutants was complemented by pSH101. The specific activity of the enzyme in cells harboring pSH101 was 37-fold higher than that in the wild type cells. gamma-Glutamyltranspeptidase was isolated from the periplasmic fraction of the cells by simple two steps and crystallized.
D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000235:genetics; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008970:Molecular Weight; D010957:Plasmids; D014169:Transformation, Bacterial; D005723:gamma-Glutamyltransferase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D004269:DNA, Bacterial; D004274:DNA, Recombinant; D005723:gamma-Glutamyltransferase; D004262:DNA Restriction Enzymes