title: Identification and characterization of polydimethylsiloxane-binding peptides (PDMS-tag) for oriented immobilization of functional protein on a PDMS surface.
abstract: In this study we focused on identifying and characterizing polydimethylsiloxane-binding peptides (PDMS-tags) that show a strong binding affinity towards a PDMS surface. Three kinds of E. coli host proteins (ELN, OMC and TPA) that were preferentially adsorbed onto a PDMS surface were identified from the E. coli cell lysate via 2-D electrophoresis and MALDI TOF MS. Digestion of these PDMS-binding proteins by 3 types of proteases (trypsin, chymotrypsin and V8 protease) resulted in the production of a wide variety of peptide fragments with different amino acid biases. Nine types of peptide fragments showing binding affinities to a PDMS surface were identified, and they were genetically fused at the C-terminal region of glutathione S-transferase (GST). The adsorption kinetics of peptide-fused GSTs to a PDMS surface were evaluated using a quartz crystal microbalance (QCM) sensor equipped with a sensor chip coated with a PDMS thin film. Consequently, all GSTs fused with the peptides adsorbed at a level higher than that of wild-type GST. In particular, the adsorption levels of GSTs fused with ELN-V81, TPA-V81, and OMC-V81 peptides were 8- to 10-fold higher than that of the wild-type GST. These results indicated that the selected peptides possessed a strong binding affinity towards a PDMS surface even in cases where they were introduced to the C-terminal region of a model protein. The remaining activities of GSTs with PDMS-binding peptides were also greater than that of the wild-type GST. Almost a third (30%) of enzymatic activity was maintained by genetic fusion of the peptide ELN-V81, compared with only 1.5% of wild-type GST in the adsorption state. Thus, the PDMS-binding peptides (PDMS-tags) identified in this study will be considerably useful for the site-specific immobilization of functional proteins to a PDMS surface, which will be a powerful tool in the fabrication of protein-based micro-reactors and biosearation chips.
authors: Department of Molecular Chemistry and Engineering, Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto, Japan. Electronic address: kumada@kit.ac.jp.
authors: Yoichi
authors: Y
authors: Kumada
authors: Department of Molecular Chemistry and Engineering, Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto, Japan.
authors: Ryoko
authors: R
authors: Otsuki
authors: Department of Molecular Chemistry and Engineering, Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto, Japan.
authors: Yumiko
authors: Y
authors: Sakoda
authors: Department of Molecular Chemistry and Engineering, Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto, Japan.
authors: Ryota
authors: R
authors: Akai
authors: Nissan Chemical Industries, Co. Ltd., Tokyo, Japan.
authors: Kazutaka
authors: K
authors: Matoba
authors: Nissan Chemical Industries, Co. Ltd., Tokyo, Japan.
authors: Junko
authors: J
authors: Katayama
authors: Department of Molecular Chemistry and Engineering, Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto, Japan.
authors: Michimasa
authors: M
authors: Kishimoto
authors: Department of Molecular Chemistry and Engineering, Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto, Japan.
authors: Jun-Ichi
authors: J
authors: Horiuchi
mesh_terms: D004129:Dimethylpolysiloxanes / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D055767:Immobilized Proteins / Q000737:chemistry* / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D004129:Dimethylpolysiloxanes; D029968:Escherichia coli Proteins; D055767:Immobilized Proteins; D010455:Peptides; C013830:baysilon
keywords: Enzyme; Material-binding peptide; Polydimethylsiloxane; Protein immobilization; Site-specific immobilization