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Cloning and overexpression of a thermostable signal peptide peptidase (SppA) from Thermoplasma volcanium GSS1 in E. coli
Author(s) -
Kocabıyık Semra,
Demirok Burçak
Publication year - 2009
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.200800344
Subject(s) - biochemistry , catalytic triad , enzyme , pmsf , signal peptide , thermophile , serine protease , biology , protease , proteases , molecular cloning , serine , peptide sequence , microbiology and biotechnology , chemistry , gene
In this study, a gene coding for thermophilic serine protease of the ClpP class from the thermoacidophilic archaeon Thermoplasma volcanium ( Tpv ) was cloned and expressed in Escherichia coli. The primary sequence and domain analysis of this enzyme showed similarities (50–60% similarity) to signal peptide peptidases (SppA) of bacteria and other archaea. An increase of about tenfold in the activity was achieved by overexpression of Tpv SppA in E. coli , as detected by enzyme assays conducted using Ala‐Ala‐Phe‐pNa and N ‐Suc‐Ala‐Ala‐Pro‐Phe‐pNA as substrates. The recombinant enzyme, purified using an anion exchange column chromatography, displayed an apparent molecular mass of 26 kDa on SDS‐PAGE analysis. Purified Tpv SppA was active in a broad range of pH and temperature with maximal activity at 60°C and between pH 7.5 and pH 8.0. The activity of the enzyme was strongly inhibited by inhibitors typical for serine proteases, i.e. , chymostatin and PMSF. The activity of the Tpv SppA and the stability at high temperature were significantly enhanced in the presence of 5 mM Ca 2+ ions. Our multiple sequence alignment data revealed a conserved Ser/Lys catalytic dyad in Tpv SppA that comprised Ser76 (nucleophile) and Lys128 (general base) residues. A search for a transmembrane domain using automated programs did not predict any signal peptide associated with the Tpv SppA and, therefore, suggested a cytoplasmic location for this enzyme.