In vitro enzymatic biotinylation of recombinant fab fragments through a peptide acceptor tail.
We describe the site-specific enzymatic biotinylation of recombinant anti-estradiol Fab fragments through a 13 amino acid acceptor peptide translationally fused to the C-terminus of the Fd chain. The Fab-peptide fusion proteins were secreted to the periplasm of Escherichia coli, purified, and biotinylated in vitro using biotin ligase, biotin, and ATP. The E. coli biotin ligase (the BirA protein) was produced as a novel N-terminal fusion protein with glutathione S-transferase (GST) and purified in one step from bacterial cell lysate using a Glutathione Sepharose affinity column. The purified fusion protein worked as such (without cleavage of the GST part) for the in vitro biotinylation of the Fab fragments. After the removal of nonbiotinylated Fab fragments by monomeric avidin chromatography, the overall yield of biotinylated Fab was 40%. The site-specifically biotinylated Fab fragments (BioFab) were tested in streptavidin-coated microtitration wells, to which they were shown to bind linearly with respect to the amount of BioFab added, specifically as indicated by biotin inhibition, and tightly with a half-life of several days. Moreover, the enzymatic BioFab exhibited uniform antigen binding affinity unlike the same recombinant Fab fragments biotinylated through random chemical conjugation to surface lysines. Finally, the BioFab demonstrated its potential as a well-behaving immunoassay reagent in a model competitive assay for estradiol.
Saviranta
P
P
Department of Biotechnology, University of Turku, Tykistökatu 6, FIN-20520 Turku, Finland. Petri.saviranta@utu.fi
Haavisto
T
T
Rappu
P
P
Karp
M
M
Lövgren
T
T
D019677:Artificial Gene Fusion; D001710:Biotin / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D004958:Estradiol / Q000737:chemistry; D007118:Immunoassay; D007120:Immunochemistry; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry*; D017879:Isothiocyanates / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry
D007140:Immunoglobulin Fab Fragments; D017879:Isothiocyanates; D010455:Peptides; D011994:Recombinant Proteins; D004958:Estradiol; D001710:Biotin