title: Solubility of disulfide-bonded proteins in the cytoplasm of Escherichia coli and its "oxidizing" mutant.
abstract: OBJECTIVE
  To study the influence of redox environment of Escherichia coli (E. coli) cytoplasm on disulfide bond formation of recombinant proteins.
  
  
  METHODS
  Bovine fibroblast growth factor (BbFGF) was selected as a model of simple proteins with a single disulfide bond and free cysteines. Anti-HBsAg single-chain Fv (HBscFv), an artificial multidomain protein, was selected as the model molecule of complex protein with 2 disulfide bonds. A BbFGF-producing plasmid, pJN-BbFGF, and a HBscFv producing-plasmid, pQE-HBscFv, were constructed and transformed into E. coli strains BL21(DE3) and M15(pREP4) respectively. At the same time, both plasmids were transformed into a reductase-deficient host strain, E. coli Origami(DE3). The 4 recombinant E. coli strains were cultured and the target proteins were purified. Solubility and bioactivity of recombinant BbFGF and HBscFv produced in different host strains were analyzed and compared respectively.
  
  
  RESULTS
  All recombinant E. coli strains could efficiently produce target proteins. The level of BbFGF in BL21(DE3) was 15-23% of the total protein, and was 5-10% in Origami (DE3). In addition, 65% of the BbFGF produced in BL21(DE3) formed into inclusion body in the cytoplasm, and all the target proteins became soluble in Origami(DE3). The bioactivity of BbFGF purified from Origami(DE3) was higher than its counterpart from BL21(DE3). The ED(50) of BbFGF from Origami(DE3) and BL21(DE3) was 1.6 microg/L and 2.2 microg/L, respectively. Both HBscFv formed into inclusion body in the cytoplasm of M15(pQE-HBscFv) or Origami(pQE-HBscFv). But the supernatant of Origami(pQE-HBscFv) lysate displayed weak bioactivity and its counterpart from M15(pQE-HBscFv) did not display any bioactivity. The soluble HBscFv in Origami(pQE-HBscFv) was purified to be 1-2 mg/L and its affinity constant was determined to be 2.62 x 10(7) mol/L. The yield of native HBscFv refolded from inclusion body in M15(pQE-HBscFv) was 30-35 mg/L and the affinity constant was 1.98 x 10(7) mol/L. There was no significant difference between the bioactivity of HBscFvs refolded from the inclusion bodies produced in different host strains.
  
  
  CONCLUSIONS
  Modification of the redox environment of E. coli cytoplasm can significantly improve the folding of recombinant disulfide-bonded proteins produced in it.
authors.affiliation: Biomedical Research and Development Center, Jinan University, Guangzhou 510630, Guangdong Province, China. xiongsheng99@263.net
authors.forename: Sheng
authors.initials: S
authors.lastname: Xiong
authors.forename: Yi-Fei
authors.initials: YF
authors.lastname: Wang
authors.forename: Xiang-Rong
authors.initials: XR
authors.lastname: Ren
authors.forename: Bing
authors.initials: B
authors.lastname: Li
authors.forename: Mei-Ying
authors.initials: MY
authors.lastname: Zhang
authors.forename: Yong
authors.initials: Y
authors.lastname: Luo
authors.forename: Ling
authors.initials: L
authors.lastname: Zhang
authors.forename: Qiu-Ling
authors.initials: QL
authors.lastname: Xie
authors.forename: Kuan-Yuan
authors.initials: KY
authors.lastname: Su
mesh_terms: D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002417:Cattle; D003593:Cytoplasm / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D009154:Mutation; D010084:Oxidation-Reduction; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism*; D012995:Solubility
chemical_list: D000906:Antibodies; D004220:Disulfides; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2
references.citation: Curr Opin Struct Biol. 2000 Aug;10(4):417-20
references.pmid: 10981628
references.citation: Proc Natl Acad Sci U S A. 1999 Nov 23;96(24):13703-8
references.pmid: 10570136
references.citation: Protein Expr Purif. 2003 Feb;27(2):267-71
references.pmid: 12597886
references.citation: Transgenic Res. 2002 Feb;11(1):61-4
references.pmid: 11874103
references.citation: Proc Natl Acad Sci U S A. 2003 Jan 21;100(2):438-42
references.pmid: 12522151
references.citation: J Immunol Methods. 1987 Jun 26;100(1-2):173-9
references.pmid: 2439600
references.citation: J Biol Chem. 1997 Jun 20;272(25):15661-7
references.pmid: 9188456
references.citation: Appl Environ Microbiol. 2004 Apr;70(4):2354-66
references.pmid: 15066832
references.citation: Sheng Wu Gong Cheng Xue Bao. 2003 Jan;19(1):19-23
references.pmid: 15969030
references.citation: J Mol Biol. 1999 Oct 1;292(4):921-9
references.pmid: 10525415
references.citation: J Immunol Methods. 1998 Oct 1;219(1-2):119-29
references.pmid: 9831393
references.citation: Mol Immunol. 2000 Dec;37(18):1123-30
references.pmid: 11451417
references.citation: J Pharmacol Toxicol Methods. 1992 Aug;28(1):9-14
references.pmid: 1392061
references.citation: Biotechnol Bioeng. 2001 Feb 5;72(3):315-22
references.pmid: 11135201
references.citation: Protein Expr Purif. 2003 Feb;27(2):272-8
references.pmid: 12597887
references.citation: Biotechnology (N Y). 1992 Apr;10(4):435-40
references.pmid: 1368488
references.citation: J Chromatogr B Biomed Sci Appl. 2000 Jan 14;737(1-2):25-38
references.pmid: 10681038
references.citation: Protein Expr Purif. 2004 May;35(1):84-92
references.pmid: 15039070
references.citation: J Mol Biol. 2002 Jun 28;320(1):1-10
references.pmid: 12079330
references.citation: World J Gastroenterol. 2004 Apr 1;10(7):977-84
references.pmid: 15052678
references.citation: Science. 2004 Feb 20;303(5661):1185-9
references.pmid: 14976313
references.citation: J Biol Chem. 2004 Jan 16;279(3):1994-2004
references.pmid: 14557270
references.citation: Gene. 1989 Jan 30;75(1):21-30
references.pmid: 2470650
references.citation: J Biol Chem. 2003 Mar 14;278(11):8979-87
references.pmid: 12519771
references.citation: J Immunol Methods. 1998 Jul 1;216(1-2):165-81
references.pmid: 9760222
references.citation: FEMS Microbiol Lett. 1998 Feb 1;159(1):41-6
references.pmid: 9485593
references.citation: Protein Expr Purif. 2000 Mar;18(2):121-32
references.pmid: 10686142
references.citation: Science. 1993 Dec 10;262(5140):1744-7
references.pmid: 8259521
references.citation: Protein Expr Purif. 2001 Jul;22(2):349-58
references.pmid: 11437612
references.citation: J Biol Chem. 2001 Apr 27;276(17):14393-9
references.pmid: 11279016
references.citation: J Biotechnol. 1999 Jun 11;72(1-2):13-20
references.pmid: 10406095
references.citation: Curr Opin Biotechnol. 1999 Oct;10(5):411-21
references.pmid: 10508629
references.citation: J Biol Chem. 1988 Dec 5;263(34):18452-8
references.pmid: 3142878
references.citation: Cell. 2004 Apr 16;117(2):199-209
references.pmid: 15084258