Expression and purification of the recombinant murine REG3α protein in Pichia pastoris and characterization of its antimicrobial and antitumour efficacy

Abstract

Regenerating islet-derived 3-alpha (REG3α) is a secreted intestinal antimicrobial protein, which shows antibacterial, anti-inflammatory and anti-apoptotic activities. It could significantly promote internal tissue regeneration and wound repair. In the present study, we expressed the codon-optimized murine REG3α in the yeast Pichia pastoris system. The secreted murine REG3α was captured using ProteinIsoTM Ni-NTA resin and further purified using a strong anion exchange resin Poros® 50 HQ. The final protein yield was 20 mg/L. The antibacterial activity and the anticancer efficacy of the recombinant REG3α were assessed using liquid growth inhibition assay, killing kinetics and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The results showed that the recombinant murine REG3α possessed antimicrobial activity against Escherichia coli, Salmonella paratyphi A, Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Bacillus pumilus and Micrococcus luteus. Moreover, 100% killing against E. coli and S. aureus was observed after 30 min. The recombinant murine REG3α had a potent antitumour activity in a time-dependent and dose-dependent manner and had a negligible haemolysis activity against human erythrocytes. Taken together, P. pastoris is an efficient expression system for producing large quantities of antibacterial active REG3α for further research studies and clinical applications.