Cloning and overexpression of a thermostable signal peptide peptidase (SppA) from Thermoplasma volcanium GSS1 in E. coli.
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 degrees 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.
Kocabiyik
Semra
S
Middle East Technical University, Department of Biological Sciences, Ankara, Turkey. biosemra@metu.edu.tr
Demirok
Burçak
B
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004492:Edetic Acid / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010802:Phylogeny; D011480:Protease Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics*; D016415:Sequence Alignment; D013822:Thermoplasma / Q000201:enzymology* / Q000235:genetics
D019843:Archaeal Proteins; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D004492:Edetic Acid; D016282:Aspartic Acid Endopeptidases; C460085:signal peptide peptidase; D002118:Calcium