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Biosynthesis of membrane dependent proteins in insect cell lysates: identification of limiting parameters for folding and processing

  • Helmut Merk EMAIL logo , Ralf-Bernhardt Rues , Christine Gless , Kerstin Beyer , Fang Dong , Volker Dötsch , Michael Gerrits and Frank Bernhard EMAIL logo
Published/Copyright: May 22, 2015
Biological Chemistry
From the journal Biological Chemistry Volume 396 Issue 9-10

Abstract

G protein-coupled receptors, like many other membrane proteins, are notoriously difficult to synthesize in conventional cellular systems. Although expression in insect cells is considered the preferred technique for structural characterizations in particular, inefficient membrane translocation, instability, toxic effects and low yields still pose clear limitations for their production in living cells. Recent studies started to explore alternative strategies for the in vitro production of problematic membrane proteins in cell-free lysates in combination with supplied membranes. We provide a detailed study on the production efficiencies and quality of G protein-coupled receptors, Fab fragments and other proteins synthesized in insect cell lysates containing endogenous microsomes. Effects of different reaction kinetics, redox conditions and sample preparations on the specific activities of synthesized proteins have been analyzed. The extent of glycosylation, membrane translocation and percentages of ligand binding active fractions of synthesized protein samples have been determined. We provide strong evidence that membrane insertion of integral membrane proteins can represent a prime limiting factor for their preparative scale in vitro production. Improved expression protocols resulting into higher production rates yielded more active protein in case of Fab fragments, but not in case of the human endothelin B receptor.

Keywords: antibodies; disulfide bridge formation; G protein-coupled receptors (GPCRs); glycosylation; ligand binding assays; membrane translocation
Corresponding authors: Helmut Merk, RiNA Netzwerk RNA-Technologien GmbH, Volmerstraße 9, D-12489 Berlin, Germany, e-mail: ; and Frank Bernhard, Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, J. W. Goethe University, Max-von-Laue Str. 9, D-60438 Frankfurt/Main, Germany, e-mail:

Acknowledgments

This study was supported by a grant from the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (BMBF, No. 0316079). Financial support was further provided by the Collaborative Research Center (SFB) 807 of the German Research Foundation (DFG). Plasmid DNA encoding ETB was kindly provided by Prof. Dr. Michael Schaefer (Universität Leipzig).

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Received: 2015-1-14
Accepted: 2015-5-11
Published Online: 2015-5-22
Published in Print: 2015-9-1

©2015 by De Gruyter

Articles in the same Issue

  1. Frontmatter
  2. Meeting Report
  3. Membrane Transport and Communication in Frankfurt: Speakers’ Summary – Highlights
  4. HIGHLIGHT: MEMBRANE TRANSPORT AND COMMUNICATION
  5. Structure, function, evolution, and application of bacterial Pnu-type vitamin transporters
  6. Team work at its best – TAPL and its two domains
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  9. Functional diversity of the superfamily of K+ transporters to meet various requirements
  10. The structure of Na+-translocating of NADH:ubiquinone oxidoreductase of Vibrio cholerae: implications on coupling between electron transfer and Na+ transport
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  12. Homeostatic control of biological membranes by dedicated lipid and membrane packing sensors
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  15. The assembly and disassembly of the AcrAB-TolC three-component multidrug efflux pump
  16. A universal mechanism for transport and regulation of CPA sodium proton exchangers
  17. Biosynthesis of membrane dependent proteins in insect cell lysates: identification of limiting parameters for folding and processing
  18. Fluorescence and excited state dynamics of the deprotonated Schiff base retinal in proteorhodopsin
  19. Regulatory role of charged clusters in the N-terminal domain of BetP from Corynebacterium glutamicum
  20. The contribution of methionine to the stability of the Escherichia coli MetNIQ ABC transporter-substrate binding protein complex
  21. The ABC exporter MsbA probed by solid state NMR – challenges and opportunities
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https://doi.org/10.1515/hsz-2015-0105
Keywords for this article
antibodies; disulfide bridge formation; G protein-coupled receptors (GPCRs); glycosylation; ligand binding assays; membrane translocation

Articles in the same Issue

  1. Frontmatter
  2. Meeting Report
  3. Membrane Transport and Communication in Frankfurt: Speakers’ Summary – Highlights
  4. HIGHLIGHT: MEMBRANE TRANSPORT AND COMMUNICATION
  5. Structure, function, evolution, and application of bacterial Pnu-type vitamin transporters
  6. Team work at its best – TAPL and its two domains
  7. The volume-regulated anion channel is formed by LRRC8 heteromers – molecular identification and roles in membrane transport and physiology
  8. Extending native mass spectrometry approaches to integral membrane proteins
  9. Functional diversity of the superfamily of K+ transporters to meet various requirements
  10. The structure of Na+-translocating of NADH:ubiquinone oxidoreductase of Vibrio cholerae: implications on coupling between electron transfer and Na+ transport
  11. Hybrid rotors in F1Fo ATP synthases: subunit composition, distribution, and physiological significance
  12. Homeostatic control of biological membranes by dedicated lipid and membrane packing sensors
  13. The transporter associated with antigen processing: a key player in adaptive immunity
  14. The pseudo-atomic structure of an RND-type tripartite multidrug efflux pump
  15. The assembly and disassembly of the AcrAB-TolC three-component multidrug efflux pump
  16. A universal mechanism for transport and regulation of CPA sodium proton exchangers
  17. Biosynthesis of membrane dependent proteins in insect cell lysates: identification of limiting parameters for folding and processing
  18. Fluorescence and excited state dynamics of the deprotonated Schiff base retinal in proteorhodopsin
  19. Regulatory role of charged clusters in the N-terminal domain of BetP from Corynebacterium glutamicum
  20. The contribution of methionine to the stability of the Escherichia coli MetNIQ ABC transporter-substrate binding protein complex
  21. The ABC exporter MsbA probed by solid state NMR – challenges and opportunities
  22. Functional properties of LptA and LptD in Anabaena sp. PCC 7120
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