title: Screening of PC and PMMA-binding peptides for site-specific immobilization of proteins.
abstract: In the present study, we used proteomic research technology to develop a method for the screening and evaluation of material-binding peptides for protein immobilization. Using this screening method, soluble Escherichia coli proteins that preferentially adsorbed onto polycarbonate (PC) and poly(methylmethacrylate) (PMMA) as model plastic materials were first isolated and identified by 2-dimensional electrophoresis (2DE) combined with peptide mass fingerprinting (PMF). The genes of identified protein candidates (ELN, MLT, OMP, and BIF) that exhibited a hexahistidine tag (6×His-tag) were over-expressed by E. coli BL21 (DE3), and the proteins were purified by IMAC affinity chromatography. The candidates for PC and PMMA-binding peptides were isolated from peptide fragments from affinity protein candidates, which were digested with trypsin and chymotrypsin. Consequently, 5 candidates for the PC-binding peptide and 2 candidates for the PMMA-binding peptide were successfully identified by MALDI-TOF MS. All of the peptides identified were introduced to the C-terminus of glutathione S-transferase (GST) as a model protein for immobilization. Adsorption of peptide-fused and wild-type GSTs onto the plastic surfaces was directly monitored using a quartz crystal microbalance (QCM) device. Consequently, genetic fusion of PC-MLT8 and PC-OMP6 as PC-binders and PM-OMP25 as a PMMA-binder significantly enhanced the adsorption rates of GST, achieving an adsorption density that was more than 10 times higher than that of wild-type GST. Furthermore, the residual activity levels of GST-PC-OMP6 and GST-PM-OMP25 in the adsorption state were 2 times higher than that of wild-type GST. Thus, the PC and PMMA-binding peptides identified in this study, namely PC-OMP6 and PM-OMP25, were considerably useful for site-specific immobilization of proteins, while maintaining a higher adsorption density and residual activity levels. The method demonstrated in this study will be applicable to the isolation of a variety of material-binding peptides against the surfaces of unique materials.
authors.affiliation: Department of Biomolecular Engineering, Kyoto Institute of Technology, Kyoto 606-8585, Japan. kumada@kit.ac.jp
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Kumada
authors.forename: Sho
authors.initials: S
authors.lastname: Murata
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Ishikawa
authors.forename: Kazuki
authors.initials: K
authors.lastname: Nakatsuka
authors.forename: Michimasa
authors.initials: M
authors.lastname: Kishimoto
mesh_terms: D000327:Adsorption; D000595:Amino Acid Sequence; D001665:Binding Sites; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods*; D010455:Peptides / Q000737:chemistry*; D011076:Polycarboxylate Cement / Q000737:chemistry*; D019904:Polymethyl Methacrylate / Q000737:chemistry*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*
chemical_list: D010455:Peptides; D011076:Polycarboxylate Cement; C005506:polycarbonate; D019904:Polymethyl Methacrylate