title: Efficient preparation and site-directed immobilization of VHH antibodies by genetic fusion of poly(methylmethacrylate)-binding peptide (PMMA-Tag).
abstract: A PMMA-binding peptide (PMMA-tag) was genetically fused with the C-terminal region of an anti-human chorionic gonadotropin (hCG) single-domain antibody (VHH). It was over-expressed in an insoluble fraction of E. coli cells, and recovered in the presence of 8 M urea via one-step IMAC purification. Monomeric and denatured PMMA-tag-fused VHH (VHH-PM) was successfully prepared via the reduction and oxidation of VHH-PM at a concentration less than 1 mg/mL in the presence of 8 M of urea. Furthermore, the VHH-PM was refolded with a recovery of more than 95% by dialysis against 50 mM TAPS at pH 8.5, because the genetic fusion of PMMA-tag resulted in a decrease in the apparent isoelectric point (pI) of the fusion protein, and its solubility at weak alkaline pH was considerably increased. The antigen-binding activities of VHH-PM in the adsorptive state were 10-fold higher than that of VHH without a PMMA-tag. The density of VHH-PM on a PMMA plate was twice that of VHH, indicating that the site-directed attachment of a PMMA-tag resulted in positive effects to the adsorption amount as well as to the orientation of VHH-PM in its adsorptive state. The preparation and immobilization methods for VHH-PM against hCG developed in the present study were further applied to VHH-PMs against four different antigens, and consequently, those antigens with the concentrations lower than 1 ng/mL were detected by the sandwich ELISA. Thus, the VHH-PMs developed in the present study are useful for preparation of high-performance and economical immunosorbent for detection of biomarkers.
authors.affiliation: Dept. of Biomolecular Engineering, Kyoto Institute of Technology, Hashigami-Cho 1, Matsugasaki, Sakyo-Ku, Kyoto, 606-8585, Japan.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Kumada
authors.affiliation: Venture Laboratory, Kyoto Institute of Technology, Hashigami-Cho 1, Matsugasaki, Sakyo-Ku, Kyoto, 606-8585, Japan.
authors.forename: Bongmun
authors.initials: B
authors.lastname: Kang
authors.affiliation: Dept. of Biomolecular Engineering, Kyoto Institute of Technology, Hashigami-Cho 1, Matsugasaki, Sakyo-Ku, Kyoto, 606-8585, Japan.
authors.forename: Kagenari
authors.initials: K
authors.lastname: Yamakawa
authors.affiliation: Dept. of Biomolecular Engineering, Kyoto Institute of Technology, Hashigami-Cho 1, Matsugasaki, Sakyo-Ku, Kyoto, 606-8585, Japan.
authors.forename: Michimasa
authors.initials: M
authors.lastname: Kishimoto
authors.affiliation: Dept. of Biomolecular Engineering, Kyoto Institute of Technology, Hashigami-Cho 1, Matsugasaki, Sakyo-Ku, Kyoto, 606-8585, Japan.
authors.forename: Jun-Ichi
authors.initials: J
authors.lastname: Horiuchi
mesh_terms: D055910:Antibodies, Immobilized / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001709:Biotechnology; D006063:Chorionic Gonadotropin; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D007526:Isoelectric Point; D019904:Polymethyl Methacrylate / Q000737:chemistry*; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility
chemical_list: D055910:Antibodies, Immobilized; D006063:Chorionic Gonadotropin; D011993:Recombinant Fusion Proteins; D061905:Single-Domain Antibodies; D019904:Polymethyl Methacrylate
keywords: ELISA; PMMA-tag; VHH; immobilization; isoelectric point; pH; refolding; solubility