Subtilisin-like serine protease from hyperthermophilic archaeon Thermococcus kodakaraensis with N- and C-terminal propeptides.
The genome of the hyperthermophilic archaeon Thermococcus kodakaraensis contains three genes encoding subtilisin-like serine proteases, Tk-1689, Tk-0076 and Tk-subtilisin. Of them, the structure and function of Tk-subtilisin have been extensively studied. To examine whether Tk-1689 is matured to an active form and functions as a hyperthermostable protease as is Tk-subtilisin, the gene encoding the Tk-1689 derivative without a putative N-terminal signal sequence, termed Pro-Tk-SP, was overexpressed in Escherichia coli. Pro-Tk-SP is composed of 640 amino acid residues and its molecular mass is 68.6 kDa. The recombinant protein was purified, however, as an active 44 kDa protease, termed Tk-SP, which lacks the N-terminal 113 and C-terminal 101 amino acid residues. This result suggests that Pro-Tk-SP consists of an N-terminal propeptide (Ala1-Ala113), a mature domain (Tk-SP, Val114-Val539) and a C-terminal propeptide (Asp540-Gly640). Like Tk-subtilisin, Tk-SP showed a broad substrate specificity and was highly thermostable. Its optimum temperature for activity was approximately 100 degrees C and its half-life at 100 degrees C was 100 min. It was fully resistant to treatment with 5% SDS, 8 M urea or 10% Triton X-100. However, unlike Tk-subtilisin and bacterial subtilisins, Tk-SP requires neither Ca2+ nor propeptide for folding. As a result, Tk-SP was fully active even in the presence of 10 mM EDTA. Thus, Tk-SP has a great advantage over other proteases in high resistance to heat, denaturants, detergents and chelating agents and therefore has great potential for application in biotechnology fields.
Foophow
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T
Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Tanaka
S
S
Koga
Y
Y
Takano
K
K
Kanaya
S
S
D000595:Amino Acid Sequence; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli; D015870:Gene Expression; D006207:Half-Life; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D057057:Serine Proteases / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity; D013381:Subtilisins / Q000235:genetics*; D013696:Temperature; D019712:Thermococcus / Q000201:enzymology*
D017931:DNA Primers; D011994:Recombinant Proteins; D057057:Serine Proteases; D013381:Subtilisins