Labeling of Cytoskeletal Proteins in Living Cells Using Biotin Ligase Carrying a Fluorescent Protein.

Labeling with fluorescent proteins is now widely exploited for elucidating the functions and roles of target proteins in living cells. Previously, we developed a protein labeling method by combining a fluorescent protein with a biotinylation reaction from archaeon Sulfolobus tokodaii. Biotinylation from S. tokodaii has a unique property that biotin protein ligase (BPL) forms a stable complex with its biotinylated substrate protein (BCCP). By taking advantage of this unique property, a target protein carrying BCCP in living cells can be labeled through biotinylation with BPL carrying a fluorescent protein. In the present work, to demonstrate the utility and performance of this labeling system in more detail, the cytoskeletal proteins ß-actin and α-tubulin were selected as target proteins and labeled in living cells. With this approach, we succeeded in fluorescent imaging of actin filaments and microtubules in living cells, and shows the advantages of our approach over the conventional labeling methods with fluorescent proteins.

Site-specific labeling of proteins by using biotin protein ligase conjugated with fluorophores.

Biotin protein ligase (BPL) mediates the covalent attachment of biotin to a specific lysine residue of biotin carboxyl carrier protein (BCCP). This biotinylation in Sulfolobus tokodaii is unique in that BPL forms a tight complex with the product, biotinylated BCCP, and this property was exploited for fluorescent labeling of a membrane protein. Thus, the truncated form of BCCP (BCCPΔ100, 69 residues) was fused to either the N or C terminus of the bradykinin B2 receptor (B2R). The resulting fusion proteins, BCCPΔ100-B2R and B2R-BCCPΔ100, respectively, were separately expressed in mammalian HEK293 cells, and labeled with BPL conjugated with a fluorophore: either fluorescein, DyLight549 or green fluorescent protein. The fusion proteins were biotinylated and bound to BPL, thereby giving rise to strong fluorescence along the periphery of the cell. Some were capable of binding bradykinin and an antagonist. When stimulated with the former, the receptor translocated to the cytosol; this suggests that the labeled receptor retains its integrity in terms of ligand-binding and translocation.