Molecular cloning of groESL locus, and purification and characterization of chaperonins, GroEL and GroES, from Bacillus brevis.
The groESL locus of a protein-hypersecreting bacterium, Bacillus brevis, was cloned by PCR using primers designed based on the DNA sequence of a B. subtilis homolog. GroEL protein was purified to apparent homogeneity and its ATPase activity was characterized: it hydrolyzed ATP, CTP, and TTP in this order of reaction rate, and its specific activity for ATP was 0.1 micromole/min/mg protein. Purified GroEL forms a tetradecamer. GroEL was estimated to contain 22% alpha-helix, 24% beta-sheet, and 19% turn structures, by CD measurement. GroES protein was also highly purified to examine its chaperonin activity. GroEL protected from thermal inactivation of and showed refolding-promoting activity for malate dehydrogenase, strictly depending on the presence of ATP and GroES.
Tokunaga
M
M
Laboratory of Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, Korimoto, Japan. tokunaga@chem.agri.kagoshima-u.ac.jp
Shiraishi
Y
Y
Odachi
M
M
Mizukami
M
M
Tokunaga
H
H
Philo
J S
JS
Arakawa
T
T
Ishibashi
M
M
Tanaka
R
R
Takagi
H
H
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001407:Bacillus / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification; D018835:Chaperonin 10 / Q000235:genetics* / Q000302:isolation & purification; D018834:Chaperonin 60 / Q000235:genetics* / Q000302:isolation & purification; D018833:Chaperonins / Q000235:genetics* / Q000302:isolation & purification; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003470:Culture Media; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D066298:In Vitro Techniques; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008929:Mitochondria, Heart / Q000378:metabolism; D018832:Molecular Chaperones / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D017510:Protein Folding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D014461:Ultracentrifugation
D001426:Bacterial Proteins; D018835:Chaperonin 10; D018834:Chaperonin 60; D003470:Culture Media; D017931:DNA Primers; D004269:DNA, Bacterial; C097970:GroESL protein, Bacteria; D018832:Molecular Chaperones; D007770:L-Lactate Dehydrogenase; D000251:Adenosine Triphosphatases; D018833:Chaperonins