Analytical Sciences

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

Hiroki MIYAO,* Utaro UEMURA,* and Shinji SUEDA*,**

*Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, 680-4 Kawazu, Iizuka 820-8502, Japan
**Research Center for Bio-microsensing Technology, Kyushu Institute of Technology, 1-1 Sensui-cho, Tobata, Kitakyushu 804-8550, Japan

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.

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