In Vitro Enzymatic Biotinylation of Recombinant Fab Fragments through a Peptide Acceptor Tail

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• 作者：Petri Saviranta ; Tapio Haavisto ; Pekka Rappu ; Matti Karp ; and Timo Lö ; vgren
• 刊名：Bioconjugate Chemistry
• 出版年：1998
• 出版时间：November 1998
• 年：1998
• 卷：9
• 期：6
• 页码：725 - 735
• 全文大小：226K
• 年卷期：v.9,no.6(November 1998)
• ISSN：1520-4812

文摘

We describe the site-specific enzymatic biotinylation of recombinant anti-estradiol Fab fragmentsthrough 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, andbiotinylated 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 purifiedin one step from bacterial cell lysate using a Glutathione Sepharose affinity column. The purifiedfusion protein worked as such (without cleavage of the GST part) for the in vitro biotinylation of theFab fragments. After the removal of nonbiotinylated Fab fragments by monomeric avidin chromatography, the overall yield of biotinylated Fab was 40%. The site-specifically biotinylated Fabfragments (BioFab) were tested in streptavidin-coated microtitration wells, to which they were shownto bind linearly with respect to the amount of BioFab added, specifically as indicated by biotininhibition, and tightly with a half-life of several days. Moreover, the enzymatic BioFab exhibiteduniform antigen binding affinity unlike the same recombinant Fab fragments biotinylated throughrandom chemical conjugation to surface lysines. Finally, the BioFab demonstrated its potential as awell-behaving immunoassay reagent in a model competitive assay for estradiol.