Cloning and overexpression of a thermostable signal peptide peptidase (SppA) from Thermoplasma volcanium GSS1 in E. coli

Abstract

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.

Microbial Diversity and Biochemical Potential Encoded by Thermal Spring Metagenomes Derived from the Kamchatka Peninsula

“…The identified serine peptidase, PepBW1, is the first metagenome-derived peptidase from a thermophilic environment. As PepBW1 is derived from an archaeal organism, it illustrates that screening in a heterologous host can be successful, even if the target gene originates from a different domain of life [56]. …”

Global role of the membrane protease LonB in Archaea: Potential protease targets revealed by quantitative proteome analysis of a lonB mutant in Haloferax volcanii

“…Consistent with the topology of bacterial SPPs, these proteins contain a signal peptide, an additional transmembrane segment and most of the protein is predicted to face the periplasm. The current knowledge on this family of proteases is limited in archaea, and only a few enzymes derived from the thermophiles have been characterized [60][61][62]. Assuming that SppA exerts similar functions in archaeal cells, it can be speculated that by affecting the levels of SppAs Lon controls signaling pathways in haloarchaea.…”

Biotechnology in Turkey: An overview

“…A line of Kocabıyık's research activities involves structure-function analysis of antibiotic resistance enzyme and thermophilic enzymes to reveal molecular mechanisms responsible for the catalysis, substrate specificity and enzyme stability. She is using a number of complementary approaches that entail cloning of appropriate genes, purification of target enzymes, rigorous characterization of their catalytic functions and probing structure-function relationships by molecular biological methods, such as site-specific mutagenesis [114][115][116][117][118]. Gülay Özcengiz has also been researching in molecular genetics [119][120][121].…”