(117b) Isolation and Characterization of Material-Binding Peptides for Site-Specific Immobilization of Proteins

 Immobilization technologies for proteins are important in the fields of bioseparation and bioanalysis. Especially, oriented immobilization of antibodies and/or their fragments onto the surfaces of solid supports such as plastic plates and chromatography resins are highly requisite for improvement of binding capacity. Affinity peptide tags such as 6xHis-tag and FLAG-tag might be useful to immobilize target proteins site-specifically onto the certain surfaces that ligand molecules are immobilized. Use of the conventional affinity peptide tags are limited because ligand molecules for capturing affinity peptide tags are sometimes not efficiently introduced to the target surfaces.

 Here, we developed material-binding peptides that directly recognize the surface structures of solid materials. By introducing material-binding peptides to target proteins, they may be site-specifically immobilized with improvement of density, orientation and remaining activity after immobilization. We developed a screening method of material-binding peptides from E. coli soluble proteins. First, protein solution extracted from E. coli cells was adsorbed onto the surface of solid materials, and the proteins that were strongly interacted with target surfaces were recovered. “The affinity proteins” that had strong affinity toward the surface of target materials were individually separated by 2D electrophoresis (2DE) and identified by peptide mass fingerprinting method utilizing MALDI-TOF MS. The affinity proteins with high purity were prepared by recombinant DNA technologies, followed by digestion with 3 types of proteases (Trypsin, Chymotrypsin and V8 protease). Binding characteristics of peptide fragments produced by proteases were analyzed by reverse-phase HPLC, and the binding peptide candidates are identified by MALDI-TOF MS and/or protein sequencer. The identified peptides were introduced to C-terminus of glutathione S-transferase as a model protein and then adsorption characteristics of peptide-tagged GSTs are further evaluated. So far, the material-binding peptides toward polystyrene (PS), polycarbonate (PC), poly(methyl methacrylate) (PMMA), silicone nitride (SiN) and other materials were successfully isolated. By introducing these peptides to antibody fragments such as single-chain Fv (scFv) antibodies, their binding capacities on the target surfaces became higher than those without peptides. Thus, the method for screening of material-binding peptides developed in this study will be useful, and other material-binding peptides will be also isolated by this method.