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

• Main Author: Yaohari, Hazarki.
• Other Authors: Susanna Leong Su Jan
• Format: Final Year Project
• Language: English
• Published: 2010
• Subjects: DRNTU::Engineering::Chemical engineering::Biotechnological production
• Online Access: http://hdl.handle.net/10356/39819
• Institution: Nanyang Technological University

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.