Pyroglutamyl-peptidase I: cloning, sequencing, and characterisation of the recombinant human enzyme

Protein Expr Purif. 2003 Mar;28(1):111-9. doi: 10.1016/s1046-5928(02)00632-0.


Pyroglutamyl-peptidase I (EC is well known from bacteria and archaea, but has not previously been cloned or sequenced from any vertebrate. We describe the cloning and sequencing of the human (AJ278828) and mouse (AJ278829) forms of pyroglutamyl-peptidase I. The deduced amino acid sequences each consist of 209 residues and show approximately 30% identity with bacterial forms of the enzyme. They show clear homology to the enzyme from prokaryotes and place the mammalian forms of the enzyme in peptidase family C15 of the MEROPS database. The catalytic residues Glu81, Cys144, and His166 in the enzyme from Bacillus amyloliquefaciens are all conserved in the human sequence. A simple cartoon model of the human protein was constructed on the basis of the published crystal structures of pyroglutamyl-peptidase I forms from Thermococcus litoralis and B. amyloliquefaciens. The human enzyme was expressed by use of a baculovirus vector in Spodoptera frugiperda cells. The recombinant protein was enzymatically active and had properties similar to those described for the naturally occurring mammalian enzyme. Gel-filtration chromatography of the active enzyme gave a molecular mass of about 24kDa, showing that the enzyme is active as the monomer. This contrasted with indications that the prokaryotic enzymes may be tetrameric. Recombinant human pyroglutamyl-peptidase I was active on pGlu-aminomethylcoumarin in the range pH 6-9, with maximal activity being seen at pH 7.0-8.5; it showed an absolute requirement for a thiol-reducing agent. In crude preparations, the enzyme was completely stable for 90 min at 50 degrees C. The enzyme was inhibited by transition metal ions including Ni(2+), Zn(2+), and Cu(2+), and by sulfhydryl-blocking agents. Reversible inhibition was seen with 2-pyrrolidone (K(i)=50 microM), and surprisingly, with N-ethylmaleimide (K(i)=30 microM).

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cloning, Molecular
  • DNA, Complementary / genetics
  • Humans
  • Hydrogen-Ion Concentration
  • Kinetics
  • Mice
  • Models, Molecular
  • Molecular Sequence Data
  • Molecular Weight
  • Protein Conformation
  • Pyroglutamyl-Peptidase I / chemistry
  • Pyroglutamyl-Peptidase I / genetics*
  • Pyroglutamyl-Peptidase I / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Analysis, DNA
  • Sequence Homology, Amino Acid
  • Temperature


  • DNA, Complementary
  • Recombinant Proteins
  • Pyroglutamyl-Peptidase I

Associated data

  • GENBANK/AJ278828
  • GENBANK/AJ278829