TY - JOUR
T1 - An extracellular halophilic protease SptA from a halophilic archaeon Natrinema sp. J7
T2 - Gene cloning, expression and characterization
AU - Shi, Wanliang
AU - Tang, Xiao Feng
AU - Huang, Yuping
AU - Gan, Fei
AU - Tang, Bing
AU - Shen, Ping
N1 - Funding Information:
Acknowledgments This work was supported in part by the National Grand Fundamental Research Program (973) of China (No.2004CB719600) and the National Natural Science Foundation of China (No.301700189, 30470033).
PY - 2006/12
Y1 - 2006/12
N2 - A gene encoding an extracellular protease, sptA, was cloned from the halophilic archaeon Natrinema sp. J7. It encoded a polypeptide of 565 amino acids containing a putative 49-amino acid signal peptide, a 103-amino acid propeptide, as well as a mature region and C-terminal extension, with a high proportion of acidic amino acid residues. The sptA gene was expressed in Haloferax volcanii WFD11, and the recombinant enzyme could be secreted into the medium as an active mature form. The N-terminal amino acid sequencing and MALDI-TOF mass spectrometry analysis of the purified SptA protease indicated that the 152-amino acid prepropeptide was cleaved and the C-terminal extension was not processed after secretion. The SptA protease was optimally active at 50°C in 2.5 M NaCl at pH 8.0. The NaCl removed enzyme retained 20% of its activity, and 60% of the activity could be restored by reintroducing 2.5 M NaCl into the NaCl removed enzyme. When the twin-arginine motif in the signal peptide of SptA protease was replaced with a twin-lysine motif, the enzyme was not exported from Hfx. volcanii WFD11, suggesting that the SptA protease was a Tat-dependent substrate.
AB - A gene encoding an extracellular protease, sptA, was cloned from the halophilic archaeon Natrinema sp. J7. It encoded a polypeptide of 565 amino acids containing a putative 49-amino acid signal peptide, a 103-amino acid propeptide, as well as a mature region and C-terminal extension, with a high proportion of acidic amino acid residues. The sptA gene was expressed in Haloferax volcanii WFD11, and the recombinant enzyme could be secreted into the medium as an active mature form. The N-terminal amino acid sequencing and MALDI-TOF mass spectrometry analysis of the purified SptA protease indicated that the 152-amino acid prepropeptide was cleaved and the C-terminal extension was not processed after secretion. The SptA protease was optimally active at 50°C in 2.5 M NaCl at pH 8.0. The NaCl removed enzyme retained 20% of its activity, and 60% of the activity could be restored by reintroducing 2.5 M NaCl into the NaCl removed enzyme. When the twin-arginine motif in the signal peptide of SptA protease was replaced with a twin-lysine motif, the enzyme was not exported from Hfx. volcanii WFD11, suggesting that the SptA protease was a Tat-dependent substrate.
KW - Extracellular protease
KW - Halophilic archaeon
KW - Natrinema sp. J7
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U2 - 10.1007/s00792-006-0003-8
DO - 10.1007/s00792-006-0003-8
M3 - Article
C2 - 16896523
AN - SCOPUS:33751107167
VL - 10
SP - 599
EP - 606
JO - Extremophiles : life under extreme conditions
JF - Extremophiles : life under extreme conditions
SN - 1431-0651
IS - 6
ER -