title: Production of recombinant disulfide-rich venom peptides for structural and functional analysis via expression in the periplasm of E. coli.
abstract: Disulfide-rich peptides are the dominant component of most animal venoms. These peptides have received much attention as leads for the development of novel therapeutic agents and bioinsecticides because they target a wide range of neuronal receptors and ion channels with a high degree of potency and selectivity. In addition, their rigid disulfide framework makes them particularly well suited for addressing the crucial issue of in vivo stability. Structural and functional characterization of these peptides necessitates the development of a robust, reliable expression system that maintains their native disulfide framework. The bacterium Escherichia coli has long been used for economical production of recombinant proteins. However, the expression of functional disulfide-rich proteins in the reducing environment of the E. coli cytoplasm presents a significant challenge. Thus, we present here an optimised protocol for the expression of disulfide-rich venom peptides in the periplasm of E. coli, which is where the endogenous machinery for production of disulfide-bonds is located. The parameters that have been investigated include choice of media, induction conditions, lysis methods, methods of fusion protein and peptide purification, and sample preparation for NMR studies. After each section a recommendation is made for conditions to use. We demonstrate the use of this method for the production of venom peptides ranging in size from 2 to 8 kDa and containing 2-6 disulfide bonds.
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Australia.
authors.forename: Julie K
authors.initials: JK
authors.lastname: Klint
authors.forename: Sebastian
authors.initials: S
authors.lastname: Senff
authors.forename: Natalie J
authors.initials: NJ
authors.lastname: Saez
authors.forename: Radha
authors.initials: R
authors.lastname: Seshadri
authors.forename: Ho Yee
authors.initials: HY
authors.lastname: Lau
authors.forename: Niraj S
authors.initials: NS
authors.lastname: Bende
authors.forename: Eivind A B
authors.initials: EA
authors.lastname: Undheim
authors.forename: Lachlan D
authors.initials: LD
authors.lastname: Rash
authors.forename: Mehdi
authors.initials: M
authors.lastname: Mobli
authors.forename: Glenn F
authors.initials: GF
authors.lastname: King
mesh_terms: D000818:Animals; D002021:Buffers; D004220:Disulfides / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D019897:Periplasm / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013329:Structure-Activity Relationship; D013696:Temperature
chemical_list: D002021:Buffers; D004220:Disulfides; D010455:Peptides; D011994:Recombinant Proteins