Overproduction and characterization of the uracil-DNA glycosylase inhibitor of bacteriophage PBS2.
A plasmid expression vector (pZWtac1) was constructed which allowed inducible overexpression of the uracil-DNA glycosylase (Ung) inhibitor (Ugi)-encoding gene (ugi) in Escherichia coli. In this plasmid, the ugi gene was under the control of both its own promoter and the tac promoter. Constitutive expression of the ugi was observed in the absence of isopropyl-beta-D-thiogalactopyranoside (IPTG). In the presence of 1 mM IPTG, the Ugi protein was overproduced to an approx. 16-fold higher level, and accounted for approx. 19% of the total soluble cellular proteins. Following high-level production in E. coli, the Ugi protein was purified to apparent homogeneity. Using E. coli Ung, we observed that Ugi inactivated the enzyme in a noncompetitive manner. Kinetic studies revealed a Ki value (0.14 microM) of approx. twelve-fold lower than Km value (1.7 microM) of glycosylase. Ugi did not act synergistically with free uracil to inhibit E. coli Ung suggesting that uracil and Ugi could share a similar mode of inhibition.
Wang
Z G
ZG
Department of Agricultural Chemistry, Oregon State University, Corvallis 97331.
Smith
D G
DG
Mosbaugh
D W
DW
D001412:Bacillus subtilis / Q000235:genetics*; D001435:Bacteriophages / Q000235:genetics*; D003001:Cloning, Molecular; D045647:DNA Glycosylases; D004926:Escherichia coli / Q000235:genetics*; D005814:Genes, Viral; D005822:Genetic Vectors; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007700:Kinetics; D009699:N-Glycosyl Hydrolases / Q000037:antagonists & inhibitors*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D015183:Restriction Mapping; D051981:Uracil-DNA Glycosidase; D014764:Viral Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology
D011994:Recombinant Proteins; D014764:Viral Proteins; C027619:uracil-DNA glycosylase inhibitor protein, B. subtilis bacteriophage; D007544:Isopropyl Thiogalactoside; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase