title: Efficient refolding and immobilization of PMMA-tag-fused single-chain Fv antibodies for sensitive immunological detection on a PMMA plate.
abstract: In this study, we investigated the efficient refolding and site-specific immobilization of single-chain variable fragments (scFvs) genetically fused with a poly(methylmethacrylate)-binding peptide (PMMA-tag). According to the results of an aggregation test of a scFv-PM in the presence of 0.5 M urea, aggregation was hardly detectable at a weak-alkaline pH (8.5) with lower concentrations of NaCl. Consequently, more than 93% recovery of the anti-RNase scFv-PM model was attained, when it was refolded by dialysis against 50 mM TAPS (pH8.5). These results suggested that the apparent isoelectric point (pI) of a target scFv was decreased to a great extent by the genetic fusion of a PMMA-tag containing 5 acidic amino acids, and, thus, the solubility of the scFv-PM in its semi-denatured form was considerably improved. We also designed alternative peptide-tags composed of plural aspartic acid residues (D5, D10 and D15-tags) to decrease the apparent pI value of the fusion protein. As a consequence, scFv-D5, scFv-D10 and scFv-D15 were also efficiently refolded with yields of more than 95%. It is noteworthy that even scFv-PS-D15, which had both a positively charged polystyrene-binding peptide (PS-tag) and a negatively charged D15-tag, was serially connected at the C-terminal region of scFvs, and also refolded with a yield of 96.1%. These results clearly indicate that controlling the apparent pI value of scFvs by the fusion of oligo-peptides composed of acidic amino acids at the C-terminus resulted in a high degree of recovery via dialysis refolding. According to the results of a sandwich ELISA using scFv-PMs, scFv-D15 and scFv-PS-D15 as ligands, high antigen-binding signals were detected from both the PMMA and phi-PS plates immobilized with scFv-PMs. Furthermore, the high antigen-binding activity of scFv-PMs was maintained in an adsorption state when it was immobilized on the surface of not only PMMA, but also hydrophilic PS (phi-PS) and polycarbonate (PC). These results strongly suggested that a PMMA-tag introduced at the C-terminus of scFvs preferably recognizes ester and/or carboxyl groups exposed on the surface of plastics. The scFv-PM developed in the present study has advantages such as being a ligand antibody, compared with whole Ab and the conventional PS-tag-fused scFvs (scFv-PS), and, thus, it is considerably useful in a sandwich ELISA as well as in various immuno-detection and immuno-separation systems.
authors.affiliation: Department of Biomolecular Engineering, Kyoto Institute of Technology, 1, Hashigami-cho, Matsugasaki, Matsugasaki, Kyoto 606-8585, Japan. Electronic address: kumada@kit.ac.jp.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Kumada
authors.affiliation: Department of Biomolecular Engineering, Kyoto Institute of Technology, 1, Hashigami-cho, Matsugasaki, Matsugasaki, Kyoto 606-8585, Japan.
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Ishikawa
authors.affiliation: Department of Biomolecular Engineering, Kyoto Institute of Technology, 1, Hashigami-cho, Matsugasaki, Matsugasaki, Kyoto 606-8585, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Fujiwara
authors.affiliation: Department of Chemistry and Materials Technology, Kyoto Institute of Technology, 1, Hashigami-cho, Matsugasaki, Matsugasaki, Kyoto 606-8585, Japan.
authors.forename: Rui
authors.initials: R
authors.lastname: Takeda
authors.affiliation: Department of Biomolecular Engineering, Kyoto Institute of Technology, 1, Hashigami-cho, Matsugasaki, Matsugasaki, Kyoto 606-8585, Japan.
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Miyamoto
authors.affiliation: Rohm Corporation, Ltd., 21 Saiin Mizosaki-cho, Ukyo-ku, Kyoto 615-8585, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Niwa
authors.affiliation: Rohm Corporation, Ltd., 21 Saiin Mizosaki-cho, Ukyo-ku, Kyoto 615-8585, Japan.
authors.forename: Shun
authors.initials: S
authors.lastname: Momose
authors.affiliation: Department of Biomolecular Engineering, Kyoto Institute of Technology, 1, Hashigami-cho, Matsugasaki, Matsugasaki, Kyoto 606-8585, Japan.
authors.forename: Bongmun
authors.initials: B
authors.lastname: Kang
authors.affiliation: Department of Biomolecular Engineering, Kyoto Institute of Technology, 1, Hashigami-cho, Matsugasaki, Matsugasaki, Kyoto 606-8585, Japan.
authors.forename: Michimasa
authors.initials: M
authors.lastname: Kishimoto
mesh_terms: D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006863:Hydrogen-Ion Concentration; D055767:Immobilized Proteins / Q000737:chemistry; D051379:Mice; D019904:Polymethyl Methacrylate / Q000737:chemistry*; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000276:immunology; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000276:immunology
chemical_list: D055767:Immobilized Proteins; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D019904:Polymethyl Methacrylate
keywords: Immobilization; Poly(methylmethacrylate)-binding peptide (PMMA-tag); Refolding; Sandwich ELISA; Single-chain Fv antibodies