Pyrrolidone Carboxyl Peptidase from the Hyperthermophilic Archaeon Pyrococcus furiosus: Cloning and Overexpression in Escherichia coli of the Gene, and Its Application to Protein Sequence Analysis

Abstract

A gene for a pyrrolidone carboxyl peptidase (Pcp: EC 3.4.19.3, pyroglutamyl peptidase), which removes amino-terminal pyroglutamyl residues from peptides and proteins, has been cloned from the hyperthermophilic Archaeon Pyrococcus furiosus using its cosmid protein library, sequenced, and expressed in Escherichia coli. The DNA sequence encodes a protein containing 208 amino acid residues with methionine at the N-terminus. Analysis of the recombinant protein expressed in E. coli, including amino acid sequence analysis from the N-terminus by automated Edman degradation and ionspray mass spectrometric analysis of the peptides generated by enzymatic digestions with lysylendopeptidase and Staphylococcus aureus V8 protease, showed its primary structure to be completely identical with that deduced from its cDNA sequence. Comparison of the amino acid sequence of P. furiosus Pcp (P.f.Pcp) with those of bacterial Pcps revealed that a high degree of sequence identity (more than 40%) and conservation of the amino acid residues comprising the catalytic triad, Cys142, His166, and Glu79. On the other hand, a unique short stretch sequence (positions around 175-185) that is absent in bacterial Pcps was found in P.f.Pcp. A similar stretch has also been reported recently in the amino acid sequence of Pcp from the hyperthermophilic Archaeon Thermococcus litoralis [Littlechild et al., in abstracts of the "International Congress on Exthermophiles '98" p. 58 (1998)]. To elucidate their contribution to the hyperthermostability of these enzymes, further structural studies are required.

Cloning and Molecular Characterization of the Basic Peroxidase Isoenzyme from Zinnia elegans, an Enzyme Involved in Lignin Biosynthesis

“…The N-terminal amino acid sequences of both ZePrx33.44 and ZePrx34.70 were determined by transferring the SDS-PAGE purified bands onto a PVDF membrane, and deblocking the N terminus with pyrrolidone carboxyl peptidase (pyroglutamyl peptidase, EC 3.4.19.3), which removes aminoterminal L-pyroglutamic acid from peptides and proteins (Hirano et al, 1993;Tsunasawa et al, 1998). For this, the PVDF membrane was excised and pretreated with 200 mL of 0.5% (w/v) polyvinylpyrrolidone-40 in 100 mM acetic acid at 37°C for 30 min to block unbound protein-binding sites on the membrane.…”

The Order Thermococcales and the Family Thermococcaceae

“…litoralis The Order Thermococcales and the Family Thermococcaceae . Tsunasawa et al (1998) Prolidase (proline dipeptidase) P. furiosus Ghosh et al (1998) P. horikoshii Theriot et al (2009Theriot et al ( , 2010 locations (see > Table 27.2). They are found all around the globe where hydrothermal are present (Lepage et al 2004).…”

Post-translational modifications of the basic peroxidase isoenzyme from Zinnia elegans

“…The N-terminal amino acid sequences of ZePrxs were determined by transferring the SDS-PAGE purified bands onto a PVDF membrane, and de-blocking the N terminus with pyrrolidone carboxyl peptidase (pyroglutamyl peptidase, EC 3.4.19.3), which removes amino-terminal L-pyroglutamic acid from peptides and proteins (Hirano et al 1993;Tsunasawa et al 1998). For this, the PVDF membrane was excised and pre-treated with 200 ll of 0.5% (w/v) polyvinylpyrrolidone-40 in 100 mM acetic acid at 37°C for 30 min to block unbound protein-binding sites on the membrane.…”