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Applied and Environmental Microbiology, April 1999, p. 1801-1805, Vol. 65, No. 4
Department of
Biotechnology1 and Department of
Material and Life Science,
Received 9 November 1998/Accepted 1 February 1999
The cpkA gene encoding a second (
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Isolation and Characterization of a Second Subunit of Molecular
Chaperonin from Pyrococcus kodakaraensis KOD1: Analysis
of an ATPase-Deficient Mutant Enzyme
) subunit of
archaeal chaperonin from Pyrococcus kodakaraensis KOD1 was
cloned, sequenced, and expressed in Escherichia coli.
Recombinant CpkA was studied for chaperonin functions in
comparison with CpkB (
subunit). The effect on decreasing the
insoluble form of proteins was examined by coexpressing CpkA or
CpkB with CobQ (cobyric acid synthase from P. kodakaraensis) in E. coli. The results indicate
that both CpkA and CpkB effectively decrease the amount of the
insoluble form of CobQ. Both CpkA and CpkB possessed the same
ATPase activity as other bacterial and eukaryal chaperonins.
The ATPase-deficient mutant proteins CpkA-D95K and
CpkB-D95K were constructed by changing conserved Asp95 to
Lys. Effect of the mutation on the ATPase activity and CobQ solubilization was examined. Neither mutant exhibited ATPase
activity in vitro. Nevertheless, they decreased the amount of the
insoluble form of CobQ by coexpression as did wild-type CpkA and CpkB.
These results implied that both CpkA and CpkB could assist protein
folding for nascent protein in E. coli without
requiring energy from ATP hydrolysis.
*
Corresponding author. Mailing address: Department of
Synthetic Chemistry and Biological Chemistry, Graduate School of
Engineering, Kyoto University, Kyoto 606-8501, Japan. Phone:
81-(0)75-753-5568. Fax: 81-(0)75-753-4703. E-mail:
imanaka{at}sbchem.kyoto-u.ac.jp.
Applied and Environmental Microbiology, April 1999, p. 1801-1805, Vol. 65, No. 4
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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