Startseite Rapid transfer of overexpressed integral membrane protein from the host membrane into soluble lipid nanodiscs without previous purification
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Rapid transfer of overexpressed integral membrane protein from the host membrane into soluble lipid nanodiscs without previous purification

  • Nazhat Shirzad-Wasei EMAIL logo , Jenny van Oostrum , Petra H.M. Bovee-Geurts , Lisanne J.A. Kusters , Giel J.C.G.M. Bosman und Willem J. DeGrip EMAIL logo
Veröffentlicht/Copyright: 10. März 2015
Biological Chemistry
Aus der Zeitschrift Biological Chemistry Band 396 Heft 8

Abstract

Structural and functional characterization of integral membrane proteins in a bilayer environment is strongly hampered by the requirement of detergents for solubilization and subsequent purification, as detergents commonly affect their structure and/or activity. Here, we describe a rapid procedure with minimal exposure to detergent to directly assemble an overexpressed integral membrane protein into soluble lipid nanodiscs prior to purification. This is exemplified with recombinant his-tagged rhodopsin, which is rapidly extracted from its host membrane and directly assembled into membrane scaffold protein (MSP) nanodiscs. We further demonstrate that, even when the MSP was his-tagged as well, partial purification of the rhodopsin-nanodiscs could be achieved exploiting immobilized-metal chromatography. Recoveries of rhodopsin up to 80% were achieved in the purified nanodisc fraction. Over 95% of contaminating membrane protein and his-tagged MSP could be removed from the rhodopsin-nanodiscs using a single Ni2+-affinity chromatography step. This level of purification is amply sufficient for functional studies. We provide evidence that the obtained rhodopsin-nanodisc preparations are fully functional both photochemically and in their ability to bind the cognate G-protein.

Keywords: baculovirus expression system; his-tagged rhodopsin; membrane protein solubilization; membrane scaffold protein; protein stabilization; recombinant protein
Corresponding authors: Nazhat Shirzad-Wasei and Willem J. DeGrip, Department of Biochemistry, 286-Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, P.O. Box 9101, NL-6500 HB Nijmegen, The Netherlands, e-mail: ;

Acknowledgments

We thank Dr. Thomas Sakmar (The Rockefeller University, New York, NY, USA) for providing the plasmid for ZAP1-expression. We acknowledge Dr. Rosalie Crouch (Medical University of South Carolina, Charleston, SC, USA) for a generous gift of 11-cis retinal through financial support from NEI. We thank Dr. Vincent Lemieux (Radboud University, Nijmegen, The Netherlands) for support with the DLS assay. This research was supported by funds to WJDG from the Council for Chemical Sciences of the Netherlands Organization for Scientific Research (NWO-CW project 700.54.008).

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Supplemental Material

The online version of this article (DOI: 10.1515/hsz-2015-0100) offers supplementary material, available to authorized users.

Received: 2015-1-3
Accepted: 2015-2-27
Published Online: 2015-3-10
Published in Print: 2015-8-1

©2015 by De Gruyter

Artikel in diesem Heft

  1. Frontmatter
  2. Reviews
  3. Ras activation revisited: role of GEF and GAP systems
  4. When core competence is not enough: functional interplay of the DEAD-box helicase core with ancillary domains and auxiliary factors in RNA binding and unwinding
  5. Cathepsin S: therapeutic, diagnostic, and prognostic potential
  6. Minireview
  7. Overview of the roles of Sox2 in stem cell and development
  8. Research Articles/Short Communications
  9. Genes and Nucleic Acids
  10. Transcriptional and translational mechanisms contribute to regulate the expression of Discs Large 1 protein during different biological processes
  11. Membranes, Lipids, Glycobiology
  12. Rapid transfer of overexpressed integral membrane protein from the host membrane into soluble lipid nanodiscs without previous purification
  13. Molecular Medicine
  14. Characterization of a new dual-targeting fully human antibody with potent antitumor activity against nasopharyngeal carcinoma
  15. Cell Biology and Signaling
  16. Lithium chloride improves the efficiency of induced pluripotent stem cell-derived neurospheres
  17. SIRT2 suppresses non-small cell lung cancer growth by targeting JMJD2A
  18. Troglitazone suppresses glutamine metabolism through a PPAR-independent mechanism
https://doi.org/10.1515/hsz-2015-0100
Schlagwörter für diesen Artikel
baculovirus expression system; his-tagged rhodopsin; membrane protein solubilization; membrane scaffold protein; protein stabilization; recombinant protein

Artikel in diesem Heft

  1. Frontmatter
  2. Reviews
  3. Ras activation revisited: role of GEF and GAP systems
  4. When core competence is not enough: functional interplay of the DEAD-box helicase core with ancillary domains and auxiliary factors in RNA binding and unwinding
  5. Cathepsin S: therapeutic, diagnostic, and prognostic potential
  6. Minireview
  7. Overview of the roles of Sox2 in stem cell and development
  8. Research Articles/Short Communications
  9. Genes and Nucleic Acids
  10. Transcriptional and translational mechanisms contribute to regulate the expression of Discs Large 1 protein during different biological processes
  11. Membranes, Lipids, Glycobiology
  12. Rapid transfer of overexpressed integral membrane protein from the host membrane into soluble lipid nanodiscs without previous purification
  13. Molecular Medicine
  14. Characterization of a new dual-targeting fully human antibody with potent antitumor activity against nasopharyngeal carcinoma
  15. Cell Biology and Signaling
  16. Lithium chloride improves the efficiency of induced pluripotent stem cell-derived neurospheres
  17. SIRT2 suppresses non-small cell lung cancer growth by targeting JMJD2A
  18. Troglitazone suppresses glutamine metabolism through a PPAR-independent mechanism
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