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Titolo:
Production of correctly folded Fab antibody fragment in the cytoplasm of Escherichia coli trxB gor mutants via the coexpression of molecular chaperones
Autore:
Levy, R; Weiss, R; Chen, G; Iverson, BL; Georgiou, G;
Indirizzi:
Univ Texas, Inst Cell & Mol Biol, Austin, TX 78712 USA Univ Texas Austin TX USA 78712 Inst Cell & Mol Biol, Austin, TX 78712 USA Univ Texas, Dept Chem Engn, Austin, TX 78712 USA Univ Texas Austin TX USA78712 exas, Dept Chem Engn, Austin, TX 78712 USA
Titolo Testata:
PROTEIN EXPRESSION AND PURIFICATION
fascicolo: 2, volume: 23, anno: 2001,
pagine: 338 - 347
SICI:
1046-5928(200111)23:2<338:POCFFA>2.0.ZU;2-O
Fonte:
ISI
Lingua:
ENG
Soggetto:
SCANNING SATURATION MUTAGENESIS; PROTEIN DISULFIDE-ISOMERASE; HIGH-LEVEL EXPRESSION; FUNCTIONAL EXPRESSION; IN-VITRO; PERIPLASMIC EXPRESSION; PHAGE DISPLAY; HIGH-AFFINITY; BINDING SITE; CODON USAGE;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
61
Recensione:
Indirizzi per estratti:
Indirizzo: Georgiou, G Univ Texas, Inst Cell & Mol Biol, Austin, TX 78712 USA Univ Texas Austin TX USA 78712 Mol Biol, Austin, TX 78712 USA
Citazione:
R. Levy et al., "Production of correctly folded Fab antibody fragment in the cytoplasm of Escherichia coli trxB gor mutants via the coexpression of molecular chaperones", PROT EX PUR, 23(2), 2001, pp. 338-347

Abstract

Disulfide bonds are normally formed after a polypeptide has been exported from the reducing environment of the cytoplasm into a more oxidizing compartment, such as the bacterial periplasm. Recently, we showed that in Escherichia coli trxB gor mutants, in which the reduction of thioredoxin and glutathione is impaired, the redox potential of the cytoplasm becomes comparableto that of the mammalian endoplasmic reticulum, thus allowing the formation of disulfide bonds in certain complex proteins (P. H. Bessette et al., 1999, Proc. Natl. Acad. Sci. USA 96,13703-13708]. Here, we investigate the expression of a Fab antibody fragment in the bacterial cytoplasm. The effect of coexpressing cytoplasmic chaperones (GroEL/ES, trigger factor, DnaK/J), as well as signal sequenceless versions of periplasmic chaperones (DsbC andSkp), was examined. Skp coexpression was shown to have the most significant effect (five- to sixfold increase) on the yield of correctly folded Fab. A maximum yield of 0.8 mg Fab/L/OD600 Fab was obtained, indicating that cytoplasmic expression may be a viable alternative for the preparative production of antibody fragments. (C) 2001 Academic Press.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 23/01/20 alle ore 03:58:08