Immobilization of immunoglobulin-G-binding domain of Protein A on a gold surface modified with biotin ligase.

Protein A from Staphylococcus aureus specifically binds to the Fc region of immunoglobulin G (IgG) and is widely used as a scaffold for the immobilization of IgG antibodies on solid supports. It is known that the oriented immobilization of Protein A on solid supports enhances its antibody-binding capability in comparison with immobilization in a random manner. In the current work, we developed a novel method for the oriented immobilization of the IgG-binding domain of Protein A based on the biotinylation reaction from archaeon Sulfolobus tokodaii. Biotinylation from S. tokodaii has a unique property in that the enzyme, biotin protein ligase (BPL), forms a stable complex with its biotinylated substrate protein, biotin carboxyl carrier protein (BCCP). Here, BCCP was fused to the IgG-binding domain of Protein A, and the resulting fusion protein was immobilized on the BPL-modified gold surface of the sensor chip for quartz crystal microbalance through complexation between BCCP and BPL. The layer of the IgG-binding domain prepared in this way successfully captured the antibody, and the captured antibody retained high antigen-binding capability.

Ultrasensitive biotin assay of a noncompetitive format in a homogeneous solution based on resonance energy transfer induced by a protein-protein interaction.

Biotin is a water-soluble vitamin serving as a cofactor for several metabolic enzymes and plays crucial roles in every living cell. In the present study, we describe a noncompetitive assay for determination of biotin in a homogeneous solution. Our assay is based on a biotinylation reaction from archaeon Sulfolobus tokodaii. S. tokodaii biotinylation has a unique property that biotin protein ligase (BPL) forms a stable complex with its biotinylated substrate protein (BCCP). Determination of biotin was performed by monitoring the complexation reaction between BPL and BCCP through biotinylation, based on luminescence resonance energy transfer (LRET) from a Tb(3+) complex to fluorescein, where BPL and BCCP were labeled with a Tb(3+) complex and fluorescein, respectively. Our assay allows for ultrasensitive detection of biotin with a detection limit of approximately 1 pM (or 0.2 fmol in a 0.2 mL sample volume) by a simple procedure without use of radioactive materials or enzymatic signal amplification. In addition, owing to its noncompetitive format, our assay has a very wide measurement range of at least 3 orders of magnitude. Our assay is also beneficial as a model system for interaction analysis based on LRET.

Stepwise Preparation of a Polymer Comprising Protein Building Blocks on a Solid Support for Immunosensing Platform.

In immunosensing, immobilization of the antibody on the sensing platform significantly influences the performance of the sensor. Herein, we propose a novel antibody-immobilization method based on a protein-polymer chain containing multiple copies of an antibody-binding protein, the Z-domain. In our approach, the Z-domain-containing polymer is prepared on the surface of the sensing platform with a biotinylation reaction from the archaeon Sulfolobus tokodaii. Biotinylation from S. tokodaii has a unique property by which biotin protein ligase (BPL) forms an extremely stable complex with its biotinylated substrate protein (BCCP). Here, we employed two types of engineered proteins: one was the fusion protein of BCCP with the Z-domain (BZB), in which BCCP was genetically attached to the N- and C-termini of the Z-domain; the other was a BPL dimer prepared by connecting two BPL molecules with a cross-linking reagent. We applied these two engineered proteins alternately onto the BPL-modified solid support of the surface plasmon resonance sensor chip, and succeeded in growing polymer chains comprising multiple units of BZB and the BPL dimer. The antibody-binding capability of the Z-domain-containing polymer thus prepared is adjustable by controlling the number of cycles of protein addition and the surface density of the polymer on the solid support.