PMID- 15742420
OWN - NLM
STAT- MEDLINE
DCOM- 20050406
LR  - 20190430
IS  - 1007-9327 (Print)
IS  - 2219-2840 (Electronic)
IS  - 1007-9327 (Linking)
VI  - 11
IP  - 7
DP  - 2005 Feb 21
TI  - Solubility of disulfide-bonded proteins in the cytoplasm of Escherichia coli and 
      its "oxidizing" mutant.
PG  - 1077-82
AB  - AIM: 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. CONCLUSION: Modification of the redox 
      environment of E. coli cytoplasm can significantly improve the folding of 
      recombinant disulfide-bonded proteins produced in it.
FAU - Xiong, Sheng
AU  - Xiong S
AD  - Biomedical Research and Development Center, Jinan University, Guangzhou 510630, 
      Guangdong Province, China. xiongsheng99@263.net
FAU - Wang, Yi-Fei
AU  - Wang YF
FAU - Ren, Xiang-Rong
AU  - Ren XR
FAU - Li, Bing
AU  - Li B
FAU - Zhang, Mei-Ying
AU  - Zhang MY
FAU - Luo, Yong
AU  - Luo Y
FAU - Zhang, Ling
AU  - Zhang L
FAU - Xie, Qiu-Ling
AU  - Xie QL
FAU - Su, Kuan-Yuan
AU  - Su KY
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - World J Gastroenterol
JT  - World journal of gastroenterology
JID - 100883448
RN  - 0 (Antibodies)
RN  - 0 (Disulfides)
RN  - 0 (Recombinant Proteins)
RN  - 103107-01-3 (Fibroblast Growth Factor 2)
SB  - IM
MH  - Animals
MH  - Antibodies/genetics/isolation & purification/metabolism
MH  - Cattle
MH  - Cytoplasm/metabolism
MH  - Disulfides/*metabolism
MH  - Escherichia coli/*genetics/*metabolism
MH  - Fibroblast Growth Factor 2/genetics/isolation & purification/metabolism
MH  - Gene Expression Regulation, Bacterial
MH  - Mutation
MH  - Oxidation-Reduction
MH  - Plasmids
MH  - Recombinant Proteins/*genetics/*metabolism
MH  - Solubility
PMC - PMC4250777
EDAT- 2005/03/03 09:00
MHDA- 2005/04/07 09:00
PMCR- 2005/02/21
CRDT- 2005/03/03 09:00
PHST- 2005/03/03 09:00 [pubmed]
PHST- 2005/04/07 09:00 [medline]
PHST- 2005/03/03 09:00 [entrez]
PHST- 2005/02/21 00:00 [pmc-release]
AID - 10.3748/wjg.v11.i7.1077 [doi]
PST - ppublish
SO  - World J Gastroenterol. 2005 Feb 21;11(7):1077-82. doi: 10.3748/wjg.v11.i7.1077.