Construction of deletion mutants of the Escherichia coli UvrA protein and their purification from inclusion bodies

L. A. Claassen, B. Ahn, Hyeon-Sook Koo, L. Grossman

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

The functions of each of the three subunits of the damage-specific UvrABC endonuclease is currently being studied by systematically mutagenizing the corresponding genes to generate mutant proteins for characterization in vitro. In this communication, we describe the construction of C-terminal deletion mutants of the UvrA protein and a procedure to purify the mutant and wild-type UvrA proteins from inclusion bodies in cells overexpressing the recombinant proteins. The method yields highly purified proteins with between 10 and 50% of the specific activity of wild-type UvrA purified by conventional techniques from the soluble fraction. The wild-type UvrA protein purified by this method had the properties of significant and selective loss of activity in assays of incision of damaged DNA, while still retaining high levels of the other unique molecular phenotypic properties associated with intact UvrA. Furthermore, the demonstration of the absolute requirement for zinc during refolding for recovery of activity is the first evidence that the zinc previously shown to be associated with the UvrA protein is in fact a necessary component for its function.

Original languageEnglish
Pages (from-to)11380-11387
Number of pages8
JournalJournal of Biological Chemistry
Volume266
Issue number17
Publication statusPublished - 1991 Sep 6

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Escherichia coli Proteins
Inclusion Bodies
Escherichia coli
Purification
Mutant Proteins
Zinc
Proteins
Endonucleases
Recombinant Proteins
Assays
Demonstrations
Genes
DNA
Recovery
Communication

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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Construction of deletion mutants of the Escherichia coli UvrA protein and their purification from inclusion bodies. / Claassen, L. A.; Ahn, B.; Koo, Hyeon-Sook; Grossman, L.

In: Journal of Biological Chemistry, Vol. 266, No. 17, 06.09.1991, p. 11380-11387.

Research output: Contribution to journalArticle

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