title: Protein complex purification from Thermoplasma acidophilum using a phage display library.
abstract: We developed a novel protein complex isolation method using a single-chain variable fragment (scFv) based phage display library in a two-step purification procedure. We adapted the antibody-based phage display technology which has been developed for single target proteins to a protein mixture containing about 300 proteins, mostly subunits of Thermoplasma acidophilum complexes. T. acidophilum protein specific phages were selected and corresponding scFvs were expressed in Escherichia coli. E. coli cell lysate containing the expressed His-tagged scFv specific against one antigen protein and T. acidophilum crude cell lysate containing intact target protein complexes were mixed, incubated and subjected to protein purification using affinity and size exclusion chromatography steps. This method was confirmed to isolate intact particles of thermosome and proteasome suitable for electron microscopy analysis and provides a novel protein complex isolation strategy applicable to organisms where no genetic tools are available.
authors.affiliation: Max-Planck Institute of Biochemistry, Department of Molecular Structural Biology, D-82152 Martinsried, Germany. Electronic address: hubert@biochem.mpg.de.
authors.forename: Agnes
authors.initials: A
authors.lastname: Hubert
authors.affiliation: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8566, Japan. Electronic address: mitani-y@aist.go.jp.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Mitani
authors.affiliation: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo 062-8517, Japan. Electronic address: t-tamura@aist.go.jp.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Tamura
authors.affiliation: Max-Planck Institute of Biochemistry, Department of Molecular Structural Biology, D-82152 Martinsried, Germany. Electronic address: boicu@biochem.mpg.de.
authors.forename: Marius
authors.initials: M
authors.lastname: Boicu
authors.affiliation: Max-Planck Institute of Biochemistry, Department of Molecular Structural Biology, D-82152 Martinsried, Germany. Electronic address: nagy@biochem.mpg.de.
authors.forename: István
authors.initials: I
authors.lastname: Nagy
mesh_terms: D001435:Bacteriophages / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D019151:Peptide Library; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D057127:Single-Chain Antibodies / Q000737:chemistry; D013822:Thermoplasma / Q000737:chemistry*
chemical_list: D019151:Peptide Library; D011506:Proteins; D057127:Single-Chain Antibodies; D046988:Proteasome Endopeptidase Complex
keywords: Electron microscopy; Phage display library; Protein complex; Thermoplasma acidophilum