In Vitro Enzymatic Biotinylation of Recombinant Fab Fragments through a Peptide Acceptor Tail Petri Saviranta,* Tapio Haavisto, Pekka Rappu, Matti Karp, and Timo Lo ¨vgren Department of Biotechnology, University of Turku, Tykisto ¨katu 6, FIN-20520 Turku, Finland. Received February 19, 1998; Revised Manuscript Received July 2, 1998 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 chroma- tography, 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. INTRODUCTION The biotin-avidin/streptavidin system is used in a variety of biotechnological and diagnostic applications owing to the exceptionally high affinity of this noncova- lent interaction between a protein and a small organic molecule (Wilchek and Bayer, 1990a). In in vitro diag- nostics, streptavidin-coated tubes, microtitration wells, or spherical particles are used as universal surfaces for the capture of biotinylated antibodies in immunoassays of various formats (Diamandis and Christopoulos, 1991). The biotinylation of antibodies can be done by several well-established chemical methods in which an activated biotin derivative is conjugated to the protein surface residues (most often lysine) to the hinge region cysteines or to a carbohydrate moeity (Bayer and Wilchek, 1990; Diamandis and Christopoulos, 1991; Hermanson, 1996). The most widely used biotin derivative, biotinyl-N- hydroxysuccinimide ester (BNHS), 1 reacts mainly with the ǫ-amino groups of surface lysines and, under optimal conditions, generally causes no serious damage to the antigen binding activity (Bayer and Wilchek, 1990). Mukkala et al. (1993) synthesized an isothiocyanate derivative of biotin (BITC), which is, like BNHS, prima- rily reactive with amino groups. The BITC reagent has since been used in our laboratory for the biotinylation of a number of different purified mAbs and has proven to be a viable complement to BNHS and other commercially available biotin reagents. The recent advances in antibody engineering (for a review, see Hayden et al., 1997) and the availability of bacterially produced recombinant Fab fragments has led to a situation in which increasingly more antibodies will be biotinylated in the Fab form. As the Fab fragment is only one-third of the size of the whole immunoglobulin, the chemical biotinylation at surface residues even at low degrees of conjugation will more often result in the destruction or modification of the antigen-binding site. The recombinant Fab fragments come with an advantage that they can be modified by protein engineering. For example, new functionalities useful in specific applica- tions can be added to the carboxyl ends of the constant domains while leaving the variable regions intact (Rap- ley, 1995). Therefore, we sought for a protein engineering solution to aid in the safe biotinylation of recombinant Fab fragments. In the living cells, a few proteins (depending on the organism) are naturally biotinylated through the covalent linkage of biotin to a unique lysine residue (Wood, 1977). This posttranslational modification is catalyzed by a biotin ligase and involves the formation of an amide bond between the carboxyl group of biotin and the ǫ-amino group of the lysine residue (Figure 4A; Shenoy and Wood, 1988). In Escherichia coli, the only biotinylated protein is the biotin carboxyl carrier protein (BCCP), a subunit of the acetyl-CoA carboxylase. The C-terminal 84-88 amino acids of BCCP form an independent domain that can be fused to recombinant proteins and biotinylated in vivo by the E. coli endogenous biotin ligase (Cronan, 1990; Li and Cronan, 1992). Although the biotinylation * Author to whom correspondence should be addressed. Tel: 358-2-333 8083. Fax: 358-2-333 8050. E-mail: Petri.saviranta@ utu.fi. 1 Abbreviations: apo-Fab, nonbiotinylated form of the Fab- biotin acceptor peptide fusion protein; BioFab, Fab fragment that is biotinylated through the 13 amino acid peptide tail; BITC, biotin isothiocyanate; BirA, the bifunctional biotin- holocarboxylase synthetase/biotin operon repressor protein of Escherichia coli; BNHS, biotinyl-N-hydroxysuccinimide ester; BSA, bovine serum albumin; dNTP, deoxynucleoside triphos- phate; GST, glutathione S-transferase of Schistosoma japoni- cum; IPTG, isopropyl -D-thiogalactopyranoside; OD600, optical density at the wavelength of 600 nm. 725 Bioconjugate Chem. 1998, 9, 725-735 10.1021/bc9800217 CCC: $15.00 © 1998 American Chemical Society Published on Web 10/03/1998