Abstract：
:Production of intracellular antibodies in Escherichia coli has been thought unlikely owing to an inability to form stable disulfide bonds in the cytoplasm, a necessary step in the folding of most immunoglobulin (Ig) domains. This work investigates whether E. coli strains carrying mutations in the major intracellular disulfide bond-reduction systems (i.e. the thioredoxin and the glutathione/glutaredoxin pathways) allow the oxidation and folding of single chain variable fragment (scFv) antibodies in the cytoplasm. The effect of the co-expression of disulfide bond chaperones in these cells was also examined. An scFv that recognizes the alternative sigma factor sigma(54) was used as a model to investigate disulfide bond formation and the folding of Ig domains in E. coli. The results demonstrate that functional intrabodies, with oxidized disulfide bonds in their Ig domains, are produced efficiently in E. coli cells carrying mutations in the glutathione oxidoreductase (gor) and the thioredoxin reductase (trxB) genes and co-expressing a signal-sequence-less derivative of the disulfide-bond isomerase DsbC ((Delta)ssDsbC). We obtained evidence indicating that (Delta)ssDsbC acts as a chaperone promoting the correct folding and oxidation of scFvs.
journal_name
J Mol Bioljournal_title
Journal of molecular biologyauthors
Jurado P,Ritz D,Beckwith J,de Lorenzo V,Fernández LAdoi
10.1016/S0022-2836(02)00405-9keywords:
subject
Has Abstractpub_date
2002-06-28 00:00:00pages
1-10issue
1eissn
0022-2836issn
1089-8638pii
S0022-2836(02)00405-9journal_volume
320pub_type
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