A biotin-based protein tagging system.
Biotin protein ligase (BPL) mediates covalent attachment of biotin to a specific lysine residue of biotin carboxyl carrier protein (BCCP) of biotin-dependent enzymes. We recently found that the biotinylation reaction from thermophilic archaeon Sulfolobus tokodaii has a unique characteristic that the enzyme BPL forms a tight complex with the product, biotinylated BCCP (169 amino acid residues). In the current work, we attempted to apply this characteristic to a novel protein tagging system. Thus, the N terminus of S. tokodaii BCCP was truncated and the interaction of the resulting BCCP, BCCPDelta100 and BCCPDelta17 (with 69 and 152 residues, respectively), with BPL was investigated by surface plasmon resonance (SPR). It was found that the binding of BPL to the biotinylated BCCPDelta100 is extremely tight with a dissociation constant (K(D)) of 1.2 nM, whereas that to the unbiotinylated counterpart was moderate with a K(D) of 3.3 microM. Furthermore, chimeric proteins of glutathione S-transferase (GST) and green fluorescence protein (GFP) with BCCPDelta100 fused to their C terminus were prepared. The resulting fusion proteins were successfully biotinylated and captured on the BPL-modified SPR sensor chip or BPL-modified magnetic beads. The function of GST and GFP was hardly impaired on fusion with BCCPDelta100 and biotinylation of the latter.
Sueda
Shinji
S
Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, Iizuka, Japan. sueda@bio.kyutech.ac.jp
Tanaka
Hitoshi
H
Yamagishi
Masanori
M
D000103:Acetyl-CoA Carboxylase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000345:Affinity Labels; D019843:Archaeal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001710:Biotin; D019921:Biotinylation / Q000379:methods*; D019731:Carbon-Nitrogen Ligases / Q000302:isolation & purification / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D054889:Fatty Acid Synthase, Type II; D017930:Genes, Reporter; D055767:Immobilized Proteins; D018189:Immunomagnetic Separation; D007700:Kinetics; D046208:Microchip Analytical Procedures; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016944:Sulfolobus / Q000201:enzymology*; D020349:Surface Plasmon Resonance
D000345:Affinity Labels; D019843:Archaeal Proteins; D002352:Carrier Proteins; D055767:Immobilized Proteins; D011993:Recombinant Fusion Proteins; D001710:Biotin; D054889:Fatty Acid Synthase, Type II; D019731:Carbon-Nitrogen Ligases; D000103:Acetyl-CoA Carboxylase; C079633:biotin carboxyl carrier protein