Molecular cloning, expression, and characterization of chaperonin-60 and chaperonin-10 from a thermophilic bacterium, Thermus thermophilus HB8.
The gene coding a chaperonin from a thermophilic bacterium, Thermus thermophilus HB8, was cloned and sequenced. The operon structure was the same as those of other bacterial chaperonins and the deduced amino acid sequences of both subunits were highly homologous to those of other chaperonins. The cloned genes of chaperonin subunits, chaperonin-10 (T.th cpn10) and chaperonin-60 (T.th cpn60), were separately expressed in Escherichia coli cells. The expressed subunits were easily purified from other host proteins including GroE, a chaperonin of E. coli. T.th cpn60 was expressed as a tetradecameric form, like GroEL of E. coli. Since chaperonin from T. thermophilus HB8 is purified as a holochaperonin, a complex of tetradecameric T.th cpn60 and heptameric T.th cpn10, a tetradecamer of T.th cpn60 without T.th cpn10 has not been obtained before. T.th cpn60 tetradecamer tended to dissociate into monomers during storage. T.th cpn10 expressed in E. coli was purified as a stable oligomer, most likely a heptamer. The activity as holo-chaperonin was reconstituted by mixing both subunits. T.th cpn60 tetradecamer itself arrested refolding of other proteins. The monomerized T.th cpn60 was easily purified from T.th cpn60 oligomer by gel permeation chromatography. Thus-obtained T.th cpn60 monomer had an ATP-independent chaperone activity, as shown for T.th cpn60 monomer isolated from authentic holo-chaperonin.
Amada
K
K
Research Laboratory of Resources Utilization, Tokyo Institute of Technology, Yokohama.
Yohda
M
M
Odaka
M
M
Endo
I
I
Ishii
N
N
Taguchi
H
H
Yoshida
M
M
D050539:3-Isopropylmalate Dehydrogenase; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D018835:Chaperonin 10 / Q000235:genetics* / Q000378:metabolism; D018834:Chaperonin 60 / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010957:Plasmids; D016963:Thermus thermophilus / Q000235:genetics*; D013884:Thiosulfate Sulfurtransferase / Q000378:metabolism
D018835:Chaperonin 10; D018834:Chaperonin 60; D004269:DNA, Bacterial; D000429:Alcohol Oxidoreductases; D050539:3-Isopropylmalate Dehydrogenase; D013884:Thiosulfate Sulfurtransferase; D000251:Adenosine Triphosphatases