ИСТИНА

The chaperonins are a class of molecular chaperones that promote protein folding in vivo and in vitro and ubiquitously found in bacteria, archaea, and eukarya. The first chaperonin GroEL ortholog encoded by gene 146 has been identified in the genome of bacteriophage EL Pseudomonas aeruginosa. Expression system of gene 146 in the E. coli cells has been developed. Purified recombinant gp146 was characterized by different physicochemical methods. Protein was found to have architecture typical for chaperonins and to consist of 14 identical subunits arranged in two heptameric rings with central cavity. The native phage substrate, gp188 or endolysin, has been immunoprecipitated from the lysate of EL-infected bacteria, using serum against recombinant gp146, and identified by mass-spectrometry. Recombinant endolysin with a 6xHis affinity tag was overexpressed in the E. coli cells in a soluble, catalytically active form. In vitro experiments have shown that the recombinant gp188 undergoes inactivation and aggregation under physiological conditions at elevated temperatures below 50 °C, while gp146 still remains native under these conditions. It was found that gp146 can protect gp188 against thermal inactivation. Using dynamic light scattering, it was demonstrated that gp146 has the capacity to suppress the irreversible aggregation of the thermally unfolded substrate molecules both in the presence and absence of ATP. The effect grows with increasing the gp146/gp188 molar ratio. However, only ATP-dependent manner seems to be similar to the chaperonin functional ATPase cycle, which results in the release of a properly folded substrate. These results provide evidence of gp146 chaperonin function. Phage chaperonin does not require co-chaperonin for its functional activity similar to group II chaperonins.