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The in vivo and in vitro characterization of DnaK from Agrobacterium tumefaciens RUOR

Boshoff, A and Hennessy, F and Blatch, Gregory L (2004) The in vivo and in vitro characterization of DnaK from Agrobacterium tumefaciens RUOR. Protein Expression and Purification, 38 (2). pp. 161-169. ISSN 1046-5928

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Abstract

Molecular chaperones of the heat shock protein 70 family (Hsp70; also called DnaK in prokaryotes) play an important role in the folding and functioning of cellular protein machinery. The dnaK gene from the plant pathogen Agrobacterium tumefaciens RUOR was amplified using the polymerase chain reaction and the DnaK protein (Agt DnaK) was over-produced as a His-tagged protein in Escherichia coli. The Agt DnaK amino acid sequence was 96% identical to the A. tumefaciens C58 DnaK sequence and 65% identical to the E. coli DnaK sequence. Agt DnaK was shown to be able to functionally replace E. coli DnaK in vivo using complementation assays with an E. coli dnaK756 mutant strain and a dnaK52 deletion strain. Over-production and purification of Agt DnaK was successful, and allowed for further characterization of the protein. Kinetic analysis of the basal ATPase activity of purified Agt DnaK revealed a Vmax of 1.3 nmol phosphate released per minute per milligram DnaK, and a Km of 62 μM ATP. Thus, this is the first study to provide both in vivo and in vitro evidence that Agt DnaK has the properties of a molecular chaperone of the Hsp70 family.

Item Type: Article
Additional Information:

Includes bibliographical references: pp. 1096-0279

Uncontrolled Keywords: ResPubID22216. Agrobacterium tumefaciens, DnaK, ATPase, complementation assays, Hsp70, protein
Subjects: Faculty/School/Research Centre/Department > School of Biomedical and Health Sciences
FOR Classification > 0601 Biochemistry and Cell Biology
FOR Classification > 0604 Genetics
Depositing User: VUIR
Date Deposited: 14 Dec 2011 06:00
Last Modified: 14 Dec 2011 06:00
URI: http://vuir.vu.edu.au/id/eprint/8161
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Citations in Scopus: 9 - View on Scopus

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