Purification and characterization of the GroESLx chaperonins from the symbiotic X-bacteria in Amoeba proteus.

GroELx and GroESx proteins of symbiotic X-bacteria from Amoeba proteus were overproduced in Escherichia coli transformed with pAJX91 and pUXGPRM, respectively, and their chaperonin functions were assayed. We utilized sigma(70)-dependent specific promoters of groEx in the expression vectors and grew recombinant cells at 37 degrees C to minimize coexpression of host groE of E. coli. For purifying the proteins, we applied the principle of heat stability for GroELx and pI difference for GroESx to minimize copurification with the hosts GroEL and GroES, respectively. After ultracentrifugation in a sucrose density gradient, the yield and purity of GroELx were 56 and 89%, respectively. The yield and purity of GroESx after anion-exchange chromatography were 62 and 91%, respectively. Purified GroELx had an ATPase activity of 53.2 nmol Pi released/min/mg protein at 37 degrees C. The GroESx protein inhibited ATPase activity of GroELx to 60% of the control at a ratio of 1 for GroESx-7mer/GroELx-14mer. GroESLx helped refolding of urea-unfolded rhodanese up to 80% of the native activity at 37 degrees C. By chemical cross-linking analysis, oligomeric properties of GroESx and GroELx were confirmed as GroESx(7) and GroELx(14) in two stacks of GroELx(7). In this study, we developed a method for the purification of GroESLx and demonstrated that their chaperonin function is homologous to GroESL of E. coli.