Abstract
We isolated and functionally characterized the α- and β- subunits (ApCpnA and ApCpnB) of a chaperonin from Aeropyrum pernix K1. The constructed vectors pET3d- ApCpnA and pET21a-ApCpnB were transformed into E. coli Rosetta (DE3), BL21 (DE3), or CodonPlus (DE3) cells. The expression of ApCpnA (60.7 kDa) and ApCpnB (61.2 kDa) was confirmed by SDS-PAGE analysis. Recombinant ApCpnA and ApCpnB were purified by heat-shock treatment and anion-exchange chromatography. ApCpnA and ApCpnB were able to hydrolyze not only ATP, but also CTP, GTP, and UTP, albeit with different efficacies. Purified ApCpnA and ApCpnB showed the highest ATPase, CTPase, UTPase, and GTPase activities at 80°C. Furthermore, the addition of ApCpnA and ApCpnB effectively protected citrate synthase (CS) and alcohol dehydrogenase (ADH) from thermal aggregation and inactivation at 43°C and 50°C, respectively. In particular, the addition of ATP or CTP to ApCpnA and ApCpnB resulted in the most effective prevention of thermal aggregation and inactivation of CS and ADH. The ATPase activity of the two chaperonin subunits was dependent on the salt concentration. Among the ions we examined, potassium ions were the most effective at enhancing the ATP hydrolysis activity of ApCpnA and ApCpnB.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Aeropyrum / enzymology*
-
Alcohol Dehydrogenase / chemistry
-
Alcohol Dehydrogenase / metabolism
-
Archaeal Proteins / chemistry
-
Archaeal Proteins / genetics
-
Archaeal Proteins / isolation & purification
-
Archaeal Proteins / metabolism*
-
Chromatography, Ion Exchange
-
Citrate (si)-Synthase / chemistry
-
Citrate (si)-Synthase / metabolism
-
Cloning, Molecular
-
Electrophoresis, Polyacrylamide Gel
-
Enzyme Stability / radiation effects
-
Escherichia coli / genetics
-
Gene Expression
-
Molecular Chaperones / chemistry
-
Molecular Chaperones / genetics
-
Molecular Chaperones / isolation & purification
-
Molecular Chaperones / metabolism*
-
Molecular Weight
-
Nucleoside-Triphosphatase / chemistry
-
Nucleoside-Triphosphatase / genetics
-
Nucleoside-Triphosphatase / isolation & purification
-
Nucleoside-Triphosphatase / metabolism
-
Protein Stability / radiation effects
-
Protein Subunits / chemistry
-
Protein Subunits / genetics
-
Protein Subunits / isolation & purification
-
Protein Subunits / metabolism
-
Recombinant Proteins / chemistry
-
Recombinant Proteins / genetics
-
Recombinant Proteins / isolation & purification
-
Recombinant Proteins / metabolism
-
Temperature
Substances
-
Archaeal Proteins
-
Molecular Chaperones
-
Protein Subunits
-
Recombinant Proteins
-
Alcohol Dehydrogenase
-
Citrate (si)-Synthase
-
Nucleoside-Triphosphatase