A plasmid expression vector (pZWtacl) 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-β-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 μM) of approx. twelve-fold lower than Km value (1.7 μM) 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.
|Number of pages||7|
|State||Published - Mar 1 1991|
Bibliographical noteFunding Information:
t We thank Xiaohua Wu for her excellentt echnical assistancea nd MatthewL ongleyf or criticale valuationo f the manuscriptW. e alsot hankD r. Bruce Duncanf or pro-vidingt hec lonedE . coli uracil-DNA glycosylaseT.h is work was supportedb y National Instituteso f Health Grant I GM32823a ndt hef ormerC laytonF oundationB iochemical + 9437f rom the OregonA griculturalE xperimentS tation. Institutea t the Universityo f Texas.T echnicalr eportN o.
- Bacillus subtilis phage
- DNA repair
- Recombinant DNA
- overexpression vector
- tac promoter
- uracil-DNA glycosylase
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