Production of a biotinylated single-chain antibody fragment in the cytoplasm of Escherichia coli.
Biotinylated antibodies are commonly used reagents in research and molecular diagnostics. The traditional approach to biotinylate antibodies is to conjugate a chemically active biotin derivative to certain chemical groups on protein surface. An alternative method, which can be used for site-specific biotinylation of recombinant antibodies, takes advantage of the capability of the enzyme biotin ligase to catalyze the attachment of a biotin to a unique lysine residue in specific protein/peptide substrates that can be genetically linked to the antibody to generate a fusion protein. We describe here expression of functional scFv and concomitant enzymatic biotinylation of it in bacterial cytoplasm. The anti-thyroid-stimulating hormone (TSH) scFv was produced as an N-terminal fusion with the biotinylated domain of the biotin carboxyl carrier protein of Escherichia coli in the redox modified E. coli strain Origami B which has an oxidizing cytoplasmic environment. After optimization of the biotin concentration and expression temperature, this approach allowed the production of biotinylated and immunoreactive fusion protein with the yield of 1.4 mg/l/OD(600) (13.6 mg/l) in a simple shake flask culture. The biotinylated fusion protein released from disrupted cells can be directly used, for example, in immunoassay applications. This was proved by setting up a TSH immunoassay using the bio-scFv as a solid-phase capture antibody. The sensitivity of the assay was comparable with the currently used commercial immunoassays.
Santala
Ville
V
Department of Biotechnology, University of Turku, FIN-20520 Turku, Finland. ville.santala@utu.fi
Lamminmäki
Urpo
U
D019921:Biotinylation / Q000379:methods; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology*; D005786:Gene Expression Regulation; D007118:Immunoassay; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012680:Sensitivity and Specificity; D013696:Temperature; D013972:Thyrotropin / Q000032:analysis
D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; D013972:Thyrotropin