Molecular Cloning of groESL Locus, and Purification and Characterization of Chaperonins, GroEL and GroES, from Bacillus brevis

Abstract

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 μmole/min/mg protein. Purified GroEL forms a tetradecamer. GroEL was estimated to contain 22% α-helix, 24% β-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.