title: Dual expression system suitable for high-throughput fluorescence-based screening and production of soluble proteins.
abstract: Many studies that aim to characterize the proteome structurally or functionally require the production of pure protein in a high-throughput format. We have developed a fast and flexible integrated system for cloning, protein expression in Escherichia coli, solubility screening and purification that can be completely automated in a 96-well microplate format. We used recombination cloning in custom-designed vectors including (i) a (His)(6) tag-encoding sequence, (ii) a variable solubilizing partner gene, (iii) the DNA sequence corresponding to the TEV protease cleavage site, (iv) the gene (or DNA fragment) of interest, (v) a suppressible amber stop codon, and (vi) an S.tag peptide-encoding sequence. First, conditions of bacterial culture in microplates (250 microL) were optimized to obtain expression and solubility patterns identical to those obtained in a 1-L flask (100-mL culture). Such conditions enabled the screening of various parameters in addition to the fusion partners (E. coli strains, temperature, inducer...). Second, expression of fusion proteins in amber suppressor strains allowed quantification of soluble and insoluble proteins by fluorescence through the detection of the S.tag. This technique is faster and more sensitive than other commonly used methods (dot blots, Western blots, SDS-PAGE). The presence of the amber suppressor tRNA was shown to affect neither the expression pattern nor the solubility of the target proteins. Third, production of the most interesting soluble fusion proteins, as detected by our screening method, could be performed in nonsuppressor strains. After cleavage with the TEV protease, the target proteins were obtained in a native form with a unique additional N-terminal glycine.
authors.affiliation: CEA/Saclay, Département d'Ingénierie et d'Etudes des Protéines, F-91191 Gif-sur-Yvette Cedex, France.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Braud
authors.forename: Mireille
authors.initials: M
authors.lastname: Moutiez
authors.forename: Pascal
authors.initials: P
authors.lastname: Belin
authors.forename: Nicolas
authors.initials: N
authors.lastname: Abello
authors.forename: Pascal
authors.initials: P
authors.lastname: Drevet
authors.forename: Sophie
authors.initials: S
authors.lastname: Zinn-Justin
authors.forename: Marie
authors.initials: M
authors.lastname: Courçon
authors.forename: Cédric
authors.initials: C
authors.lastname: Masson
authors.forename: Janie
authors.initials: J
authors.lastname: Dassa
authors.forename: Jean-Baptiste
authors.initials: JB
authors.lastname: Charbonnier
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Boulain
authors.forename: André
authors.initials: A
authors.lastname: Ménez
authors.forename: Roger
authors.initials: R
authors.lastname: Genet
authors.forename: Muriel
authors.initials: M
authors.lastname: Gondry
mesh_terms: D015153:Blotting, Western; D003001:Cloning, Molecular; D018388:Codon, Terminator; D004247:DNA / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry; D005786:Gene Expression Regulation; D015964:Gene Expression Regulation, Bacterial; D016153:Genes, Suppressor; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D011506:Proteins / Q000737:chemistry; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D012343:RNA, Transfer / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013696:Temperature
chemical_list: D018388:Codon, Terminator; D029968:Escherichia coli Proteins; D011506:Proteins; D020543:Proteome; D011993:Recombinant Fusion Proteins; D004247:DNA; D012343:RNA, Transfer; D005982:Glutathione Transferase