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 comparable to 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 and Skp), 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/OD(600) Fab was obtained, indicating that cytoplasmic expression may be a viable alternative for the preparative production of antibody fragments.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Levy R,Weiss R,Chen G,Iverson BL,Georgiou Gdoi
10.1006/prep.2001.1520keywords:
subject
Has Abstractpub_date
2001-11-01 00:00:00pages
338-47issue
2eissn
1046-5928issn
1096-0279pii
S1046-5928(01)91520-7journal_volume
23pub_type
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
pub_type: 杂志文章
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journal_title:Protein expression and purification
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