Abstract
Filamentous bacteriophages have been used in numerous applications for the display of antibodies and random peptide libraries. Here we describe the introduction of a 13 amino acid sequence LASIFEAQKIEWR (designated BT, which is biotinylated in vivo by E. coli) into the N termini of four of the five structural proteins of the filamentous bacteriophage fd (Proteins 3, 7, 8 and 9). The in vivo and in vitro biotinylation of the various phages were compared. The production of multifunctional phages and their application as affinity reagents are demonstrated.
Highlights
Biotinylation of proteins, nucleic acids, lipids and sugars is without a doubt one of the most fundamental tools of modern cell biology and biotechnology
biotin carboxyl carrier protein (BCCP) is biotinylated on the epsilon amine of lysine 122 via an amide bond formed at the expense of ATP [7,8], a reaction that is catalyzed by the biotin holoenzyme synthetase
Schatz [13] has demonstrated that a 13-15aa peptidomimetic of the lysine-containing turn can be produced that is efficiently recognized by Biotin holoenzyme synthetase (BHS) and can be used as a biotinylation tag (BT) either N’ terminal or C’ terminal to recombinant fusion proteins expressed in E. coli
Summary
Biotinylation of proteins, nucleic acids, lipids and sugars is without a doubt one of the most fundamental tools of modern cell biology and biotechnology. This is due to the scarcity of naturally biotinylated proteins (< 5 per organism) [1,2,3], the chemical flexibility by which biotin can be covalently conjugated to specific moieties of biopolymers and organic ligands, and the exceptional high affinity binding between avidin/streptavidin and biotin (KD = 10-14M) [4]. In E. coli biotin carboxyl carrier protein (BCCP) is the only protein that is biotinylated and is one of four subunits of the enzyme acetyl CoA carboxylase which is functional in fatty acid biosynthesis. Schatz [13] has demonstrated that a 13-15aa peptidomimetic of the lysine-containing turn can be produced that is efficiently recognized by BHS and can be used as a biotinylation tag (BT) either N’ terminal or C’ terminal to recombinant fusion proteins expressed in E. coli (available as a commercial biotinylation tag coined “Avi-Tag”, GeneCopoeia, Inc., Rockville)
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