Cloning, expression, and sequencing of a protease gene (tpr) from Porphyromonas gingivalis W83 in Escherichia coli

Infect Immun. 1992 Aug;60(8):3186-92. doi: 10.1128/iai.60.8.3186-3192.1992.

Abstract

Porphyromonas gingivalis is a highly proteolytic organism which metabolizes small peptides and amino acids. Indirect evidence suggests that the proteases produced by this microorganism constitute an important virulence factor. In this study, a gene bank of P. gingivalis W83 DNA was constructed by cloning 0.5- to 20-kb HindIII-cut DNA fragments into Escherichia coli DH5 alpha by using the plasmid vector pUC19. A clone expressing a protease from P. gingivalis was isolated on LB agar containing 1% skim milk. The clone contained a 3.0-kb insert that coded for a protease with an apparent molecular mass of 64 kDa. Sequencing part of the 3.0-kb DNA fragment revealed an open reading frame encoding a protein of 482 amino acids with a molecular mass of 62.5 kDa. Putative promoter and termination elements flanking the open reading frame were identified. The activity expressed in E. coli was extensively characterized by using various substrates and protease inhibitors, and the results suggest that it is possibly a thiol protease.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Cloning, Molecular*
  • Endopeptidases / genetics*
  • Escherichia coli / genetics*
  • Genes, Bacterial*
  • Hydrogen-Ion Concentration
  • Molecular Sequence Data
  • Porphyromonas gingivalis / enzymology
  • Porphyromonas gingivalis / genetics*
  • Protease Inhibitors / pharmacology
  • Restriction Mapping

Substances

  • Protease Inhibitors
  • Endopeptidases

Associated data

  • GENBANK/M24252
  • GENBANK/M27037
  • GENBANK/M27305
  • GENBANK/M27306
  • GENBANK/M63610
  • GENBANK/M63611
  • GENBANK/M63612
  • GENBANK/M63613
  • GENBANK/M63614
  • GENBANK/M63615
  • GENBANK/M63616
  • GENBANK/M63617
  • GENBANK/M84471
  • GENBANK/X03970
  • GENBANK/X63185
  • GENBANK/Y00641
  • GENBANK/Y00708