Cloning and expression of the 5'-nucleotidase gene of Vibrio parahaemolyticus in Escherichia coli and overproduction of the enzyme.
The gene encoding the membrane-bound 5'-nucleotidase of Vibrio parahaemolyticus was cloned and expressed in Escherichia coli. Cells of E. coli harboring a plasmid, pNUT5, which carries the 5'-nucleotidase gene were able to grow on ATP as the sole source of carbon, although the original cells were not. The 5'-nucleotidase activity was detected in whole cells of E. coli harboring pNUT5 and in membrane vesicles prepared from these cells. Most properties of the 5'-nucleotidase produced in E. coli, that is, its requirements for Cl- and Mg2+, substrate specificity, and inhibition by Zn2+, were similar to those observed in V. parahaemolyticus, but some alterations in properties were observed: The 5'-nucleotidase was partially inducible in V. parahaemolyticus, but its expression in E. coli was completely constitutive. The specific activity of the 5'-nucleotidase in membrane vesicles of E. coli harboring the plasmid was 30 times that observed in whole cells, whereas the specific activities in membrane vesicles and in whole cells of V. parahaemolyticus were almost the same. A new, dense band of protein with an apparent molecular mass of 63 kDa was detected when membrane proteins of E. coli harboring the plasmid were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Sakai
Y
Y
Department of Microbiology, Faculty of Pharmaceutical Sciences, Okayama University.
Tamao
Y
Y
Shimamoto
T
T
Hama
H
H
Tsuda
M
M
Tsuchiya
T
T
D015720:5'-Nucleotidase; D015139:Blotting, Southern; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D009708:Nucleotidases / Q000096:biosynthesis* / Q000235:genetics; D013379:Substrate Specificity; D014736:Vibrio parahaemolyticus / Q000201:enzymology* / Q000235:genetics
D004269:DNA, Bacterial; D004262:DNA Restriction Enzymes; D009708:Nucleotidases; D015720:5'-Nucleotidase