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J Microbiol Methods. 2014 Mar;98:15-22. doi: 10.1016/j.mimet.2013.12.010. Epub 2013 Dec 21.

Protein complex purification from Thermoplasma acidophilum using a phage display library.

Author information

  • 1Max-Planck Institute of Biochemistry, Department of Molecular Structural Biology, D-82152 Martinsried, Germany. Electronic address: hubert@biochem.mpg.de.
  • 2Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8566, Japan. Electronic address: mitani-y@aist.go.jp.
  • 3Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo 062-8517, Japan. Electronic address: t-tamura@aist.go.jp.
  • 4Max-Planck Institute of Biochemistry, Department of Molecular Structural Biology, D-82152 Martinsried, Germany. Electronic address: boicu@biochem.mpg.de.
  • 5Max-Planck Institute of Biochemistry, Department of Molecular Structural Biology, D-82152 Martinsried, Germany. Electronic address: nagy@biochem.mpg.de.

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.

Copyright © 2013 Elsevier B.V. All rights reserved.

KEYWORDS:

Electron microscopy; Phage display library; Protein complex; Thermoplasma acidophilum

PMID:
24365107
[PubMed - indexed for MEDLINE]
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