Molecular cloning of the Lon protease gene from Thermus thermophilus HB8 and characterization of its gene product

The gene encoding Lon protease was isolated from an extreme thermophile, Th ermus thermophilus HB8. Sequence analysis demonstrated that the T. thermoph ilus Lon protease gene (TT-lon) contains a protein-coding sequence consisti ng of 2385 bp which is approximate to 56% homologous to the Escherichia col i counterpart. As expected, the G/C content of TT-lon was 68%, which is sig nificantly higher than that of the E, coli ion gene (52% G/C). The amino ac id sequence of T. thermophilus Lon protease (TT-Lon) predicted from the nuc leotide sequence contained several unique sequences conserved in other Lon proteases: (a) a cysteine residue at the position just before the putative ATP-binding domain; (b) motif A and B sequences required for composition of the ATP-binding domain; and (c) a serine residue at the proteolytic active site. Expression of TT-lon under the control of the T7 promoter in E. coli produced an 89-kDa protein with a yield of approximate to 5 mg.L-1. Recomb inant TT-Lon (rTT-Lon) was purified to homogeneity by sequential column chr omatography. The peptidase activity of rTT-Lon was activated by ATP and alp ha-casein. rTT-Lon cleaved succinyl-phenylalanyl-leucyl-phenylalanyl-methox ynaphthylamide much more efficiently than succinyl-alanyl-alanyl-phenylalan yl-methoxynaphthylamide, whereas both peptides were cleaved with comparable efficiencies by E. coli Lon. These results suggest that there is a differe nce between TT-Lon and E. coli Lon in substrate specificity. rTT-Lon most e ffectively cleaved substrate peptides at 70 degrees C, which was significan tly higher than the optimal temperature (37 degrees C) for E. coli Lon. Tog ether, these results indicate that the TT-lon? gene isolated from;T: thermo philus HB8 actually encodes an ATP-dependent thermostable protease Lon.