Heterologous Expression, Purification and Characterization of an Oligopeptidase A from the Pathogen Leptospira interrogans

• Published in: Molecular biotechnology 2018-04, Vol.60 (4), p.302-309
• Main Authors: Anu, Prasannan V., Madanan, Madathiparambil G., Nair, Ananthakrishnan J., Nair, Gangaprasad A., Nair, Govinda Pillai M., Sudhakaran, Perumana R., Satheeshkumar, Padikara K.
• Format: Article
• Language: English
• Subjects: Affinity chromatography; Amino acids; Biochemistry; Biological Techniques; Biotechnology; Cell Biology; Chemistry; Chemistry and Materials Science; Cysteine proteinase; Diagnostic systems; E coli; Histidine; Human Genetics; Leptospira; Leptospira interrogans; Leptospirosis; Membrane proteins; Oligopeptidase; Oligopeptidase A; Original Paper; Overexpression; Pathogens; Peptidase; Peptides; Protease; Protease inhibitors; Protein Science; Proteinase inhibitors; Proteins; Proteomes; Salmonella; Serine; Serine proteinase; Viability; Virulence; Virulence factors; Zinc
• ISSN: 1073-6085; 1559-0305
• DOI: 10.1007/s12033-018-0073-8

Oligopeptidases are enzymes involved in the degradation of short peptides (generally less than 30 amino acids in size) which help pathogens evade the host defence mechanisms. Leptospira is a zoonotic pathogen and causes leptospirosis in mammals. Proteome analysis of Leptospira revealed the presence of oligopeptidase A (OpdA) among other membrane proteins. To study the role of oligopeptidase in leptospirosis, the OpdA of L. interrogans was cloned and expressed in Escherichia coli with a histidine tag (His-tag). The protein showed maximum expression at 37 °C with 0.5 mM of IPTG after 2 h of induction. Recombinant OpdA protein was purified to homogeneity using Ni-affinity chromatography. The purified OpdA showed more than 80% inhibition with a serine protease inhibitor but the activity was reduced to 30% with the cysteine protease inhibitor. The peptidase activity was increased significantly in the presence of Zn 2+ at a neutral pH. Inhibitor assay indicate the presence of more than one active sites for peptidase activity as reported with the OpdA of E. coli and Salmonella. Over-expression of OpdA in E. coli BL21 (DE3) did not cause any negative effects on normal cell growth and viability. The role of OpdA as virulence factor in Leptospira and its potential as a therapeutic and diagnostic target in leptospirosis is yet to be identified.