In vitro stabilization and in vivo solubilization of foreign proteins by the beta subunit of a chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1.
The gene encoding the beta subunit of a molecular chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1 (cpkB) was cloned, sequenced, and expressed in Escherichia coli. The cpkB gene is composed of 1,641 nucleotides, encoding a protein (546 amino acids) with a molecular mass of 59,140 Da. The enhancing effect of CpkB on enzyme stability was examined by using Saccharomyces cerevisiae alcohol dehydrogenase (ADH). Purified recombinant CpkB prevents thermal denaturation and enhances thermostability of ADH. CpkB requires ATP for its chaperonin function at a low CpkB concentration; however, CpkB functions without ATP when present in excess. In vivo chaperonin function for the solubilization of insoluble proteins was also studied by coexpressing CpkB and CobQ (cobryic acid synthase), indicating that CpkB is useful for solubilizing the insoluble proteins in vivo. These results suggest that the beta subunit plays a major role in chaperonin activity and is functional without the alpha subunit.
Yan
Z
Z
Department of Biotechnology, Graduate School of Engineering, Osaka University, Japan.
Fujiwara
S
S
Kohda
K
K
Takagi
M
M
Imanaka
T
T
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000426:Alcohol Dehydrogenase / Q000187:drug effects*; D000595:Amino Acid Sequence; D001105:Archaea / Q000235:genetics*; D019843:Archaeal Proteins; D001483:Base Sequence; D018833:Chaperonins / Q000235:genetics* / Q000494:pharmacology*; D003001:Cloning, Molecular; D004795:Enzyme Stability / Q000187:drug effects; D006358:Hot Temperature; D008969:Molecular Sequence Data; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D011994:Recombinant Proteins / Q000187:drug effects; D017422:Sequence Analysis, DNA; D012995:Solubility; D000637:Transaminases / Q000187:drug effects*
D019843:Archaeal Proteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D000426:Alcohol Dehydrogenase; D000637:Transaminases; C071151:cobyric acid synthase; D018833:Chaperonins