Functional antibody single-chain fragments from the cytoplasm of Escherichia coli: influence of thioredoxin reductase (TrxB).
The cytoplasmic expression of a functional antibody (Ab) fragment, containing the correct intradomain disulfide bonds, was investigated in E. coli. We used a single-chain Fv (scFv) fragment of the levan-binding Ab ABPC48, which was shown to be functional only in the presence of the disulfide bonds. Significant amounts of functional, disulfide-containing scFv could be produced in the cytoplasm of E. coli in the absence of thioredoxin reductase (TrxB) activity. The amount of soluble protein remained largely unchanged by this null mutation. A stronger promoter did not result in further improved yields of functional Ab fragment, despite much higher protein production, suggesting that inefficient disulfide formation was still limiting the yield of active scFv. This method of expressing functional Ab fragments in the cytoplasm of E. coli may be important for screening and selection systems.
Proba
K
K
Biochemisches Institut, Universität Zürich, Switzerland.
Ge
L
L
Plückthun
A
A
D000937:Antigen-Antibody Reactions; D017123:Bacteriophage T7 / Q000235:genetics; D004220:Disulfides / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D005630:Fructans / Q000276:immunology*; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis; D013329:Structure-Activity Relationship; D013880:Thioredoxin-Disulfide Reductase / Q000235:genetics / Q000378:metabolism*
D004220:Disulfides; D005630:Fructans; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; D013880:Thioredoxin-Disulfide Reductase