title: High throughput quantitative expression screening and purification applied to recombinant disulfide-rich venom proteins produced in E. coli.
abstract: Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4 ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment. Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (www.venomics.eu), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques (AFMB), Aix-Marseille Université
authors.forename: Natalie J
authors.initials: NJ
authors.lastname: Saez
authors.affiliation: iBiTec-S, Service d'Ingénierie Moléculaire des Protéines (SIMOPRO), Commissariat à l'énergie atomique et aux énergies alternatives (CEA) Saclay, France.
authors.forename: Hervé
authors.initials: H
authors.lastname: Nozach
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques (AFMB), Aix-Marseille Université
authors.forename: Marilyne
authors.initials: M
authors.lastname: Blemont
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques (AFMB), Aix-Marseille Université; Renaud.Vincentelli@afmb.univ-mrs.fr.
authors.forename: Renaud
authors.initials: R
authors.lastname: Vincentelli
mesh_terms: D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D019704:Protein Disulfide-Isomerases / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D014688:Venoms / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D014688:Venoms; D019704:Protein Disulfide-Isomerases; C526649:dsbC protein, E coli