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

• Published in: Protein expression and purification Vol. 38; no. 2; pp. 161 - 169
• Main Authors: Boshoff, Aileen, Hennessy, Fritha, Blatch, Gregory L.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.12.2004
• Subjects: Adenosine Triphosphatases - metabolism; Adenosine Triphosphate - metabolism; Agrobacterium tumefaciens; Agrobacterium tumefaciens - genetics; Amino Acid Sequence; ATPase; Bacterial Proteins - biosynthesis; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Complementation assays; DnaK; Escherichia coli Proteins - genetics; Gene Expression Regulation, Bacterial; Genetic Complementation Test; Hsp70; HSP70 Heat-Shock Proteins - biosynthesis; HSP70 Heat-Shock Proteins - genetics; HSP70 Heat-Shock Proteins - isolation & purification; Hydrolysis; Molecular Sequence Data; Sequence Homology, Amino Acid; Agrobacterium tumefaciens; Complementation assays; Hsp70; DnaK; ATPase
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2004.06.039

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 V max of 1.3 nmol phosphate released per minute per milligram DnaK, and a K m 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.