title: High throughput screening identifies disulfide isomerase DsbC as a very efficient partner for recombinant expression of small disulfide-rich proteins in E. coli.
abstract: BACKGROUND
  Disulfide-rich proteins or DRPs are versatile bioactive compounds that encompass a wide variety of pharmacological, therapeutic, and/or biotechnological applications. Still, the production of DRPs in sufficient quantities is a major bottleneck for their complete structural or functional characterization. Recombinant expression of such small proteins containing multiple disulfide bonds in the bacteria E. coli is considered difficult and general methods and protocols, particularly on a high throughput scale, are limited.
  
  
  RESULTS
  Here we report a high throughput screening approach that allowed the systematic investigation of the solubilizing and folding influence of twelve cytoplasmic partners on 28 DRPs in the strains BL21 (DE3) pLysS, Origami B (DE3) pLysS and SHuffle® T7 Express lysY (1008 conditions). The screening identified the conditions leading to the successful soluble expression of the 28 DRPs selected for the study. Amongst 336 conditions tested per bacterial strain, soluble expression was detected in 196 conditions using the strain BL21 (DE3) pLysS, whereas only 44 and 50 conditions for soluble expression were identified for the strains Origami B (DE3) pLysS and SHuffle® T7 Express lysY respectively. To assess the redox states of the DRPs, the solubility screen was coupled with mass spectrometry (MS) to determine the exact masses of the produced DRPs or fusion proteins. To validate the results obtained at analytical scale, several examples of proteins expressed and purified to a larger scale are presented along with their MS and functional characterization.
  
  
  CONCLUSIONS
  Our results show that the production of soluble and functional DRPs with cytoplasmic partners is possible in E. coli. In spite of its reducing cytoplasm, BL21 (DE3) pLysS is more efficient than the Origami B (DE3) pLysS and SHuffle® T7 Express lysY trxB(-)/gor(-) strains for the production of DRPs in fusion with solubilizing partners. However, our data suggest that oxidation of the proteins occurs ex vivo. Our protocols allow the production of a large diversity of DRPs using DsbC as a fusion partner, leading to pure active DRPs at milligram scale in many cases. These results open up new possibilities for the study and development of DRPs with therapeutic or biotechnological interest whose production was previously a limitation.
authors.affiliation: CEA, iBiTec-S, Service d'Ingénierie Moléculaire des Protéines, CEA Saclay, Gif sur Yvette F-91191, France. herve.nozach@cea.fr
authors.forename: Hervé
authors.initials: H
authors.lastname: Nozach
authors.forename: Carole
authors.initials: C
authors.lastname: Fruchart-Gaillard
authors.forename: François
authors.initials: F
authors.lastname: Fenaille
authors.forename: Fabrice
authors.initials: F
authors.lastname: Beau
authors.forename: Oscar Henrique Pereira
authors.initials: OH
authors.lastname: Ramos
authors.forename: Badreddine
authors.initials: B
authors.lastname: Douzi
authors.forename: Natalie J
authors.initials: NJ
authors.lastname: Saez
authors.forename: Mireille
authors.initials: M
authors.lastname: Moutiez
authors.forename: Denis
authors.initials: D
authors.lastname: Servent
authors.forename: Muriel
authors.initials: M
authors.lastname: Gondry
authors.forename: Robert
authors.initials: R
authors.lastname: Thaï
authors.forename: Philippe
authors.initials: P
authors.lastname: Cuniasse
authors.forename: Renaud
authors.initials: R
authors.lastname: Vincentelli
authors.forename: Vincent
authors.initials: V
authors.lastname: Dive
mesh_terms: D003593:Cytoplasm / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D004220:Disulfides; D011993:Recombinant Fusion Proteins; D019704:Protein Disulfide-Isomerases