title: A screening strategy for heterologous protein expression in Escherichia coli with the highest return of investment.
abstract: Heterologous protein expression in Escherichia coli is commonly used to obtain recombinant proteins for a variety of downstream applications. However, many proteins are not, or are only poorly, expressed in soluble form. High level expression often leads to the formation of inclusion bodies and an inactive product that needs to be refolded. By screening the solubility pattern for a set of 71 target proteins in different host-strains and varying parameters such as location of purification tag, promoter and induction temperature we propose a protocol with a success rate of 77% of clones returning a soluble protein. This protocol is particularly suitable for high-throughput screening with the goal to obtain soluble protein product for e.g. structure determination.
authors.affiliation: Structural Genomics Consortium, University of Toronto, MaRS South Tower, Suite 700, 101 College St., Toronto, ON, Canada.
authors.forename: Benny
authors.initials: B
authors.lastname: Pacheco
authors.affiliation: Structural Genomics Consortium, University of Toronto, MaRS South Tower, Suite 700, 101 College St., Toronto, ON, Canada.
authors.forename: Lissete
authors.initials: L
authors.lastname: Crombet
authors.affiliation: Structural Genomics Consortium, University of Toronto, MaRS South Tower, Suite 700, 101 College St., Toronto, ON, Canada.
authors.forename: Peter
authors.initials: P
authors.lastname: Loppnau
authors.affiliation: Structural Genomics Consortium, University of Toronto, MaRS South Tower, Suite 700, 101 College St., Toronto, ON, Canada. Electronic address: doug.cossar@utoronto.ca.
authors.forename: Doug
authors.initials: D
authors.lastname: Cossar
mesh_terms: D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006639:Histidine; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012995:Solubility; D013696:Temperature
chemical_list: C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine