A Strategy for Production of Correctly Folded Disulfide-Rich Peptides in the Periplasm of E. coli

• DOI: 10.1007/978-1-4939-6887-9_10
• Publication Date: 2017-05-03T04:49:17Z
• Authors: Natalie J. Saez; Ben Cristofori-Ar...; Raveendra Anangi; Glenn F. King
• Keywords: Disulfide-rich protein; Protein Folding; Disulfide-rich peptide (DRP); Recombinant Fusion Proteins; E. coli; Liquid chromatography; TEV protease cleavage; Chromatography, Affinity; 3. Good health; Recombinant expression; Transformation, Genetic; 1311 Genetics; Solubility; Periplasm; Venom peptide; 1312 Molecular Biology; Escherichia coli; Electrophoresis, Polyacrylamide Gel; Disulfides; Peptides; Purification; Chromatography, High Pressure Liquid; Plasmids

Recombinant expression of disulfide-reticulated peptides and proteins is often challenging. We describe a method that exploits the periplasmic disulfide-bond forming machinery of Escherichia coli and combines this with a cleavable, solubility-enhancing fusion tag to obtain higher yields of correctly folded target protein than is achievable via cytoplasmic expression. The protocols provided herein cover all aspects of this approach, from vector construction and transformation to purification of the cleaved target protein and subsequent quality control.