Enfuvirtide biosynthesis in thermostable chaperone-based fusion

Abstract

Synthetic peptides are in high demand as biologically active substances. Solid phase synthesis is the primary method of peptide production. However, it has drawbacks: large amount of chemical waste and rapid increase in price with peptide length. Biosynthesis is intended as method to bypass these flaws. Direct biosynthesis is usually not effective and among other approaches for improving quality and quantity of target product fusion partners are widely used. In this study we used a thermostable chaperon-based fusion partner developed by us to produce enfuvirtide in Escherichia coli expression system. Fusion partner's thermal stability provided additional purification mode by thermal denaturation of host proteins in lysate. Fusion protein was purified by ion exchange chromatography after lysate heating step and was then hydrolyzed with cyanogen bromide to release enfuvirtide. Enfuvirtide was isolated by RP-HPLC up to 94% purity with total yield of 2.86–3.31 mg per 1 L of low-density culture. The data demonstrate the posibility of thermostable chaperone-based fusion partner GroEL use for effective peptide biosynthesis.

Keywords

• Molecular chaperones
• Fusion system
• Peptide biosynthesis
• Protein engineering
• Thermostable protein