title: Expression of eukaryotic proteins in soluble form in Escherichia coli.
abstract: At the optimum temperature for its growth (37 degrees C), Escherichia coli tends to accumulate heterologous proteins in insoluble form. Fusion protein technology has been used to increase the solubility of overexpressed proteins in this organism, but with variable degrees of success. Fusion to a mutant form of DsbA (DsbAmut) confers higher levels of solubility to heterologous proteins in a reproducible way, even when E. coli is grown at 37 degrees C. We have shown this to be true with a diverse sample of eukaryotic proteins: IGF-I, IGFBP-3, 3C proteinase, TGF beta-2, sTGF beta-RII, BDNF, GDNF, mEGFBP, leptin, and GFP. In addition, we have investigated the effects of charge average and proline content on the solubility of DsbAmut fusions. Coexpression of a protein prolyl isomerase [cyclophilin (L-)] and modification of selected asparagine residues to aspartic acid appear to have beneficial effects on the accumulation of soluble heterologous proteins.
authors.affiliation: Department of Molecular and Cell Biology, Celtrix Pharmaceuticals, Inc., Santa Clara, California 95054, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Zhang
authors.forename: D R
authors.initials: DR
authors.lastname: Olsen
authors.forename: K B
authors.initials: KB
authors.lastname: Nguyen
authors.forename: P S
authors.initials: PS
authors.lastname: Olson
authors.forename: E T
authors.initials: ET
authors.lastname: Rhodes
authors.forename: D
authors.initials: D
authors.lastname: Mascarenhas
mesh_terms: D001483:Base Sequence; D019208:Brain-Derived Neurotrophic Factor / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005057:Eukaryotic Cells / Q000737:chemistry; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D051100:Glial Cell Line-Derived Neurotrophic Factor; D049452:Green Fluorescent Proteins; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D020738:Leptin; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D019696:Peptidylprolyl Isomerase / Q000235:genetics; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D017346:Protein Serine-Threonine Kinases; D011506:Proteins / Q000737:chemistry / Q000235:genetics; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D012995:Solubility; D016212:Transforming Growth Factor beta / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D019208:Brain-Derived Neurotrophic Factor; D002352:Carrier Proteins; D017931:DNA Primers; C495039:GDNF protein, human; D051100:Glial Cell Line-Derived Neurotrophic Factor; D018972:Insulin-Like Growth Factor Binding Protein 3; D020738:Leptin; D008164:Luminescent Proteins; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D011506:Proteins; D018125:Receptors, Transforming Growth Factor beta; D011993:Recombinant Fusion Proteins; D016212:Transforming Growth Factor beta; D049452:Green Fluorescent Proteins; D007334:Insulin-Like Growth Factor I; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II; D003546:Cysteine Endopeptidases; D019696:Peptidylprolyl Isomerase