Characterization of three putative Lon proteases of Thermus thermophilus HB27 and use of their defective mutants as hosts for production of heterologous proteins |
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Authors: | Tomoko Maehara Takayuki Hoshino Akira Nakamura |
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Institution: | (1) Division of Integrative Environmental Sciences, Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba Ibaraki, 305-8572, Japan |
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Abstract: | In the genome of a thermophilic bacterium, Thermus thermophilus HB27, three genes, TTC0418, TTC0746 and TTC1975, were annotated as ATP-dependent protease La (Lon). Sequence comparisons indicated that TTC0418 and TTC0746 showed significant
similarities to bacterial LonA-type proteases, such as Escherichia coli Lon protease, especially in regions corresponding to domains for ATP-binding and hydrolysis, and for proteolysis, but TTC1975
exhibited a similarity only at the C-terminal proteolytic domain. The enzymatic analyses, using purified recombinant proteins
produced by E. coli, revealed that TTC0418 and TTC0746 exhibited peptidase and protease activities against two synthetic peptides and casein,
respectively, in an ATP-dependent manner, and at the same time, both the enzymes had significant ATPase activities in the
presence of substrates. On the other hand, TTC1975 possessed a protease activity against casein, but addition of ATP did not
enhance this activity. Moreover, a T. thermophilus mutant deficient in both TTC0418 and TTC0746 showed a similar growth characteristic to an E. coli lon mutant, i.e., a growth defect lag after a nutritional downshift. These results indicate that TTC0418 and TTC0746 are actually
members of bacterial LonA-type proteases with different substrate specificities, whereas TTC1975 should not be classified
as a Lon protease. Finally, the effects of mutations deficient in these proteases were assessed on production of several heterologous
gene products from Pyrococcus horikoshii and Geobacillus stearothermophilus. It was shown that TTC0746 mutation was more effective in improving production than the other two mutations, especially for production of P. horikoshii α-mannosidase and G. stearothermophilus α-amylase, indicating that the TTC0746 mutant of T. thermophilus HB27 may be useful for production of heterologous proteins from thermophiles and hyperthermophiles. |
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Keywords: | Thermus thermophilus Lon protease Protease/peptidase ATPase Heterologous gene expression |
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