title: Versatile C-terminal specific biotinylation of proteins using both a puromycin-linker and a cell-free translation system for studying high-throughput protein-molecule interactions.
abstract: Immobilization of a protein in a functionally active form and correct orientation for high-throughput analysis is crucial for surface-based protein-molecular interaction studies and should aid progress in associated nanotechnologies. Here, we present a general method for controlled and oriented immobilization of proteins by a puromycin-linker for cDNA display technology. The utility and potential of this method was demonstrated by examining the interaction between the B domain of protein A and immunoglobulin G (IgG) by surface plasmon resonance. This study revealed that the mRNA fragment of the mRNA-protein fusion (i.e., mRNA display) interferes with the interaction between the protein (B domain) and its target molecule (IgG). This results in a reduction of the apparent affinity by ~10-fold. This method is expected to find wide appeal in the fields of surface-based studies of protein-protein interactions, drug screening, and single molecule analysis that require only a small amount of protein sample.
authors: Graduate School of Science and Engineering, Saitama University , 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.
authors: Naoto
authors: N
authors: Nemoto
authors: Takayuki
authors: T
authors: Fukushima
authors: Shigefumi
authors: S
authors: Kumachi
authors: Miho
authors: M
authors: Suzuki
authors: Koichi
authors: K
authors: Nishigaki
authors: Tai
authors: T
authors: Kubo
mesh_terms: D001710:Biotin / Q000737:chemistry* / Q000378:metabolism; D019921:Biotinylation; D002474:Cell-Free System; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism; D014176:Protein Biosynthesis; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D011691:Puromycin / Q000737:chemistry*; D058525:Quartz Crystal Microbalance Techniques; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000737:chemistry* / Q000378:metabolism; D020349:Surface Plasmon Resonance
chemical_list: D055767:Immobilized Proteins; D007074:Immunoglobulin G; D012333:RNA, Messenger; D013205:Staphylococcal Protein A; D011691:Puromycin; D001710:Biotin