Biosynthesis of membrane dependent proteins in insect cell lysates: identification of limiting parameters for folding and processing
-
Helmut Merk
, Ralf-Bernhardt Rues
, Christine Gless , Kerstin Beyer , Fang Dong , Volker Dötsch , Michael Gerrits und Frank Bernhard
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.
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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©2015 by De Gruyter
Artikel in diesem Heft
- Frontmatter
- Meeting Report
- Membrane Transport and Communication in Frankfurt: Speakers’ Summary – Highlights
- HIGHLIGHT: MEMBRANE TRANSPORT AND COMMUNICATION
- Structure, function, evolution, and application of bacterial Pnu-type vitamin transporters
- Team work at its best – TAPL and its two domains
- The volume-regulated anion channel is formed by LRRC8 heteromers – molecular identification and roles in membrane transport and physiology
- Extending native mass spectrometry approaches to integral membrane proteins
- Functional diversity of the superfamily of K+ transporters to meet various requirements
- The structure of Na+-translocating of NADH:ubiquinone oxidoreductase of Vibrio cholerae: implications on coupling between electron transfer and Na+ transport
- Hybrid rotors in F1Fo ATP synthases: subunit composition, distribution, and physiological significance
- Homeostatic control of biological membranes by dedicated lipid and membrane packing sensors
- The transporter associated with antigen processing: a key player in adaptive immunity
- The pseudo-atomic structure of an RND-type tripartite multidrug efflux pump
- The assembly and disassembly of the AcrAB-TolC three-component multidrug efflux pump
- A universal mechanism for transport and regulation of CPA sodium proton exchangers
- Biosynthesis of membrane dependent proteins in insect cell lysates: identification of limiting parameters for folding and processing
- Fluorescence and excited state dynamics of the deprotonated Schiff base retinal in proteorhodopsin
- Regulatory role of charged clusters in the N-terminal domain of BetP from Corynebacterium glutamicum
- The contribution of methionine to the stability of the Escherichia coli MetNIQ ABC transporter-substrate binding protein complex
- The ABC exporter MsbA probed by solid state NMR – challenges and opportunities
- Functional properties of LptA and LptD in Anabaena sp. PCC 7120
Artikel in diesem Heft
- Frontmatter
- Meeting Report
- Membrane Transport and Communication in Frankfurt: Speakers’ Summary – Highlights
- HIGHLIGHT: MEMBRANE TRANSPORT AND COMMUNICATION
- Structure, function, evolution, and application of bacterial Pnu-type vitamin transporters
- Team work at its best – TAPL and its two domains
- The volume-regulated anion channel is formed by LRRC8 heteromers – molecular identification and roles in membrane transport and physiology
- Extending native mass spectrometry approaches to integral membrane proteins
- Functional diversity of the superfamily of K+ transporters to meet various requirements
- The structure of Na+-translocating of NADH:ubiquinone oxidoreductase of Vibrio cholerae: implications on coupling between electron transfer and Na+ transport
- Hybrid rotors in F1Fo ATP synthases: subunit composition, distribution, and physiological significance
- Homeostatic control of biological membranes by dedicated lipid and membrane packing sensors
- The transporter associated with antigen processing: a key player in adaptive immunity
- The pseudo-atomic structure of an RND-type tripartite multidrug efflux pump
- The assembly and disassembly of the AcrAB-TolC three-component multidrug efflux pump
- A universal mechanism for transport and regulation of CPA sodium proton exchangers
- Biosynthesis of membrane dependent proteins in insect cell lysates: identification of limiting parameters for folding and processing
- Fluorescence and excited state dynamics of the deprotonated Schiff base retinal in proteorhodopsin
- Regulatory role of charged clusters in the N-terminal domain of BetP from Corynebacterium glutamicum
- The contribution of methionine to the stability of the Escherichia coli MetNIQ ABC transporter-substrate binding protein complex
- The ABC exporter MsbA probed by solid state NMR – challenges and opportunities
- Functional properties of LptA and LptD in Anabaena sp. PCC 7120