The in vivo and in vitro characterization of DnaK from Agrobacterium tumefaciens RUOR.
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 microM 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.
Boshoff
Aileen
A
Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, Grahamstown 6140, South Africa.
Hennessy
Fritha
F
Blatch
Gregory L
GL
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D016960:Agrobacterium tumefaciens / Q000235:genetics*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D029968:Escherichia coli Proteins / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D006868:Hydrolysis; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli