Mo } ecular C } oning of groESL Locus , of Chaperonins , GroEL and

The groESL locus of a protein-hypersecreting bacterium, BaciULts 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 O.lpmole!minfmg protein. Purified GroEL forms a tetradecamer. GroEL was estimated to contain 22% orhelix, 24% fi-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-