title: Manipulating the aggregation and oxidation of human SPARC in the cytoplasm of Escherichia coli.
abstract: Human SPARC (secreted protein acidic and rich in cysteine), an extracellular matrix protein containing 14 cysteine residues, was found to partition equally between soluble and insoluble cellular fractions when overexpressed in the Escherichia coli cytoplasm. While the growth temperature and medium pH had little effect on inclusion body formation, co-overproduction of the dnaKJ operon, but not of the groE operon, suppressed aggregation at the expense of intracellular accumulation. Although both forms of the protein were fully reduced in wild-type cells, 70% to 85% of soluble and insoluble SPARC could be converted into oxidized species in a thioredoxin reductase (trxB) null mutant following incubation on ice. Approximately 15% to 20% of SPARC exhibited the electrophoretic mobility of the biologically active protein. Overproduction of the dnaKJ operon in trxB cells decreased the formation of disulfide-bonded SPARC multimers in the aggregated material but not in its soluble counterpart. Our results suggest that the activity responsible for disulfide bond formation in trxB mutants acts at the post-translational level and is able to freely diffuse within inclusion bodies.
authors.affiliation: Department of Chemical Engineering, University of Washington, Seattle 98195, USA.
authors.forename: E L
authors.initials: EL
authors.lastname: Schneider
authors.forename: J G
authors.initials: JG
authors.lastname: Thomas
authors.forename: J A
authors.initials: JA
authors.lastname: Bassuk
authors.forename: E H
authors.initials: EH
authors.lastname: Sage
authors.forename: F
authors.initials: F
authors.lastname: Baneyx
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular / Q000379:methods; D003545:Cysteine; D003593:Cytoplasm / Q000378:metabolism; D004220:Disulfides / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation; D006801:Humans; D009876:Operon; D015676:Osteonectin / Q000096:biosynthesis / Q000378:metabolism*; D010084:Oxidation-Reduction; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism
chemical_list: D004220:Disulfides; D015676:Osteonectin; D011994:Recombinant Proteins; D003545:Cysteine