Molecular cloning of the Lon protease gene from Thermus thermophilus HB8 and characterization of its gene product
1999
Watanabe, Satoru | Muramatsu, Tomonari | Ao, Hiroko | Hirayama, Yoshie | Takahashi, Kenji | Tanokura, Masaru | Kuchino, Yoshiyuki
The gene encoding Lon protease was isolated from an extreme thermophile, Thermus thermophilus HB8. Sequence analysis demonstrated that the T. thermophilus Lon protease gene (TT‐lon) contains a protein‐coding sequence consisting of 2385 bp which is ≈ 56% homologous to the Escherichia coli counterpart. As expected, the G/C content of TT‐lon was 68%, which is significantly higher than that of the E. coli lon gene (52% G/C). The amino acid sequence of T. thermophilus Lon protease (TT‐Lon) predicted from the nucleotide 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 ≈ 5 mg·L⁻¹. Recombinant TT‐Lon (rTT‐Lon) was purified to homogeneity by sequential column chromatography. The peptidase activity of rTT‐Lon was activated by ATP and α‐casein. rTT‐Lon cleaved succinyl‐phenylalanyl‐leucyl‐phenylalanyl‐methoxynaphthylamide much more efficiently than succinyl‐alanyl‐alanyl‐phenylalanyl‐methoxynaphthylamide, whereas both peptides were cleaved with comparable efficiencies by E. coli Lon. These results suggest that there is a difference between TT‐Lon and E. coli Lon in substrate specificity. rTT‐Lon most effectively cleaved substrate peptides at 70 °C, which was significantly higher than the optimal temperature (37 °C) for E. coli Lon. Together, these results indicate that the TT‐lon gene isolated from T. thermophilus HB8 actually encodes an ATP‐dependent thermostable protease Lon.
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