Optimization of tobacco etch virus (TEV) protease cleavage in fusion proteins.

Abstract

This report summarizes the study of tobacco etch virus protease (TEVp) cleavage efficiency to enhance the productivity of antimicrobial peptide human beta-defensin (HBD) 25, a promising new class of antibiotics. Four parameters were investigated: salt concentration, substrate to enzyme ratio, cleavage time and pH. To avoid antimicrobial activity towards the host, the HBD 25 was expressed as a soluble fusion with maltose-binding protein (MBP) in Escherichia coli. Luria-Bertani (LB) was used as the culture medium for E. coli BL21 (DE3). The MBP-HBD25 fusion protein was then cleaved by the TEVp digestion enzyme at room temperature (25°C) in various conditions. The highest yield of HBD25 target peptide was obtained after 6 h of cleavage when 50 mM Tris-HCl, 2 M urea (pH = 8) digestion buffer was used, with 5:1 substrate to enzyme weight ratio. An HBD25 concentration of 0.032 g/l in the cleavage product solution was achieved with the mentioned cleavage condition. The cleavage recovery and yield were 83% and 11% (g HBD25/g substrate), respectively. Importantly, HBD25 yield was decreased by increasing salt (NaCl) concentration.