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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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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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Abbreviations

AAA+:

ATPase associated with various cellular activities

ORF:

Open reading frame

IPTG:

Isopropyl-β-d-thiogalactopyranoside

EcLon:

Lon protease of E. coli

TtLon:

Lon protease of T. thermophilus

SDS-PAGE:

SDS-polyacrylamide gel electrophoresis

5-FOA:

5-Fluoroorotic acid

Glt-AAF-MNA:

Glutaryl-Ala-Ala-Phe-methoxynaphthylamide

Suc-FLF-MNA:

Succinyl-Phe-Leu-Phe-methoxynaphthylamide

Thr-DH:

Threonine dehydrogenase

α-Man:

α-Mannosidase

Glu-DH:

Glutamate dehydrogenase

α-amy:

α-Amylase

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Authors and Affiliations

  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

    Tomoko Maehara, Takayuki Hoshino & Akira Nakamura

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  1. Tomoko Maehara
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  2. Takayuki Hoshino
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  3. Akira Nakamura
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Correspondence to Akira Nakamura.

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Communicated by K. Horikoshi.

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Maehara, T., Hoshino, T. & Nakamura, A. Characterization of three putative Lon proteases of Thermus thermophilus HB27 and use of their defective mutants as hosts for production of heterologous proteins. Extremophiles 12, 285–296 (2008). https://doi.org/10.1007/s00792-007-0129-3

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  • DOI: https://doi.org/10.1007/s00792-007-0129-3

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