title: Soluble expression, purification and functional characterization of a coil peptide composed of a positively charged and hydrophobic motif.
abstract: A de novo heterodimeric coiled-coil system formed by the association of two synthetic peptides, the Ecoil and Kcoil, has been previously designed and proven to be an excellent and versatile tool for various biotechnology applications. However, based on the challenges encountered during its chemical synthesis, the Kcoil peptide has been designated as a "difficult peptide". In this study, we explore the expression of the Kcoil peptide by a bacterial system as well as its subsequent purification. The maximum expression level was observed when the peptide was fused to thioredoxin and the optimized purification process consisted of three chromatographic steps: immobilized-metal affinity chromatography followed by cation-exchange chromatography and, finally, a reverse-phase high-performance liquid chromatography. This entire process led to a final volumetric production yield of 1.5 mg of pure Kcoil peptide per liter of bacterial culture, which represents a significant step towards the cost-effective production and application of coiled-coil motifs. Our results thus demonstrate for the first time that bacterial production is a viable alternative to the chemical synthesis of de novo designed coil peptides.
authors.affiliation: Department of Chemical Engineering, Groupe de Recherche en Sciences et Technologies Biomédicales (GRSTB), Bio-P² Research Unit, École Polytechnique de Montréal, P.O. Box 6079, Succ. Centre-Ville, Montreal, QC, H3C 3A7, Canada.
authors.forename: Nesrine
authors.initials: N
authors.lastname: Riahi
authors.affiliation: Department of Biochemistry, Université de Montréal, PO Box 6128, Succ. Centre-Ville, Montreal, QC, H3C 3J7, Canada.
authors.forename: Laurent
authors.initials: L
authors.lastname: Cappadocia
authors.affiliation: Department of Chemical Engineering, Groupe de Recherche en Sciences et Technologies Biomédicales (GRSTB), Bio-P² Research Unit, École Polytechnique de Montréal, P.O. Box 6079, Succ. Centre-Ville, Montreal, QC, H3C 3A7, Canada.
authors.forename: Olivier
authors.initials: O
authors.lastname: Henry
authors.affiliation: Department of Biochemistry, Université de Montréal, PO Box 6128, Succ. Centre-Ville, Montreal, QC, H3C 3J7, Canada.
authors.forename: James
authors.initials: J
authors.lastname: Omichinski
authors.affiliation: Department of Chemical Engineering, Groupe de Recherche en Sciences et Technologies Biomédicales (GRSTB), Bio-P² Research Unit, École Polytechnique de Montréal, P.O. Box 6079, Succ. Centre-Ville, Montreal, QC, H3C 3A7, Canada. gregory.decrescenzo@polymtl.ca.
authors.forename: Gregory
authors.identifier: http://orcid.org/0000-0002-6280-1570
authors.initials: G
authors.lastname: De Crescenzo
mesh_terms: D020816:Amino Acid Motifs; D060326:Chemistry Techniques, Synthetic / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D010452:Peptide Biosynthesis / Q000502:physiology*; D010455:Peptides / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013879:Thioredoxins / Q000378:metabolism
chemical_list: D010455:Peptides; D013879:Thioredoxins
keywords: Bacterial production; Coil peptide; Soluble expression; Thioredoxin