Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 85, Issue 2, Pages 218-223Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2012.08.008
Keywords
Bacteriophage T7; T7 protein kinase; Autophosphorylation; IF1; Ribonuclease III
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Funding
- National Institutes of Health [RO1 GM56772]
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Bacteriophage T7 encodes a serine/threonine-specific protein kinase that phosphorylates multiple cellular proteins during infection of Escherichia coli. Recombinant 17 protein kinase (T7PK), normally purified in phosphorylated form, exhibits a modest level of phosphotransferase activity. A procedure is described that provides dephosphorylated T7PK with an enhanced ability to phosphorylate protein substrates, including translation initiation factor IF1 and the nuclease domain of ribonuclease III. Mass spectrometric analysis identified Thr12 as the site of IF1 phosphorylation in vitro. T7PK undergoes Mg2+-dependent autophosphorylation on Ser216 in vitro, which also is modified in vivo. The inability to isolate the presumptive autophosphorylation-resistant T7PK Ser216Ala mutant indicates a toxicity of the phosphotransferase activity and suggests a role for Ser216 modification in limiting T7PK activity during infection. (C) 2012 Elsevier Inc. All rights reserved.
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