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Title: Cell-Free Co-Translational Approaches for Producing Mammalian Receptors: Expanding the Cell-Free Expression Toolbox Using Nanolipoproteins

Abstract

Membranes proteins make up more than 60% of current drug targets and account for approximately 30% or more of the cellular proteome. Access to this important class of proteins has been difficult due to their inherent insolubility and tendency to aggregate in aqueous solutions. Understanding membrane protein structure and function demands novel means of membrane protein production that preserve both their native conformational state as well as function. Over the last decade, cell-free expression systems have emerged as an important complement to cell-based expression of membrane proteins due to their simple and customizable experimental parameters. One approach to overcome the solubility and stability limitations of purified membrane proteins is to support them in stable, native-like states within nanolipoprotein particles (NLPs), aka nanodiscs. This has become common practice to facilitate biochemical and biophysical characterization of proteins of interest. NLP technology can be easily coupled with cell-free systems to achieve functional membrane protein production for this purpose. Our approach involves utilizing cell-free expression systems in the presence of NLPs or using co-translation techniques to perform one-pot expression and self-assembly of membrane protein/NLP complexes. We describe how cell-free reactions can be modified to render control over nanoparticle size and monodispersity in support ofmore » membrane protein production. These modifications have been exploited to facilitate co-expression of full-length functional membrane proteins such as G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). In particular, we summarize the state of the art in NLP-assisted cell-free coexpression of these important classes of membrane proteins as well as evaluate the advances in and prospects for this technology that will drive drug discovery against these targets. We conclude with a prospective on the use of NLPs to produce as well as deliver functional mammalian membrane-bound proteins for a range of applications.« less

Authors:
; ; ; ;
Publication Date:
Research Org.:
Lawrence Livermore National Laboratory (LLNL), Livermore, CA (United States)
Sponsoring Org.:
USDOE National Nuclear Security Administration (NNSA)
OSTI Identifier:
1530895
Alternate Identifier(s):
OSTI ID: 1557951
Report Number(s):
LLNL-JRNL-768744
Journal ID: ISSN 1663-9812; 744
Grant/Contract Number:  
AC52-07NA27344
Resource Type:
Published Article
Journal Name:
Frontiers in Pharmacology
Additional Journal Information:
Journal Name: Frontiers in Pharmacology Journal Volume: 10; Journal ID: ISSN 1663-9812
Publisher:
Frontiers Media SA
Country of Publication:
Switzerland
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; cell-free; co-translation; nanolipoprotein particle; nanodisc; membrane proteins

Citation Formats

Shelby, Megan L., He, Wei, Dang, Amanda T., Kuhl, Tonya L., and Coleman, Matthew A.. Cell-Free Co-Translational Approaches for Producing Mammalian Receptors: Expanding the Cell-Free Expression Toolbox Using Nanolipoproteins. Switzerland: N. p., 2019. Web. doi:10.3389/fphar.2019.00744.
Shelby, Megan L., He, Wei, Dang, Amanda T., Kuhl, Tonya L., & Coleman, Matthew A.. Cell-Free Co-Translational Approaches for Producing Mammalian Receptors: Expanding the Cell-Free Expression Toolbox Using Nanolipoproteins. Switzerland. https://doi.org/10.3389/fphar.2019.00744
Shelby, Megan L., He, Wei, Dang, Amanda T., Kuhl, Tonya L., and Coleman, Matthew A.. Wed . "Cell-Free Co-Translational Approaches for Producing Mammalian Receptors: Expanding the Cell-Free Expression Toolbox Using Nanolipoproteins". Switzerland. https://doi.org/10.3389/fphar.2019.00744.
@article{osti_1530895,
title = {Cell-Free Co-Translational Approaches for Producing Mammalian Receptors: Expanding the Cell-Free Expression Toolbox Using Nanolipoproteins},
author = {Shelby, Megan L. and He, Wei and Dang, Amanda T. and Kuhl, Tonya L. and Coleman, Matthew A.},
abstractNote = {Membranes proteins make up more than 60% of current drug targets and account for approximately 30% or more of the cellular proteome. Access to this important class of proteins has been difficult due to their inherent insolubility and tendency to aggregate in aqueous solutions. Understanding membrane protein structure and function demands novel means of membrane protein production that preserve both their native conformational state as well as function. Over the last decade, cell-free expression systems have emerged as an important complement to cell-based expression of membrane proteins due to their simple and customizable experimental parameters. One approach to overcome the solubility and stability limitations of purified membrane proteins is to support them in stable, native-like states within nanolipoprotein particles (NLPs), aka nanodiscs. This has become common practice to facilitate biochemical and biophysical characterization of proteins of interest. NLP technology can be easily coupled with cell-free systems to achieve functional membrane protein production for this purpose. Our approach involves utilizing cell-free expression systems in the presence of NLPs or using co-translation techniques to perform one-pot expression and self-assembly of membrane protein/NLP complexes. We describe how cell-free reactions can be modified to render control over nanoparticle size and monodispersity in support of membrane protein production. These modifications have been exploited to facilitate co-expression of full-length functional membrane proteins such as G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). In particular, we summarize the state of the art in NLP-assisted cell-free coexpression of these important classes of membrane proteins as well as evaluate the advances in and prospects for this technology that will drive drug discovery against these targets. We conclude with a prospective on the use of NLPs to produce as well as deliver functional mammalian membrane-bound proteins for a range of applications.},
doi = {10.3389/fphar.2019.00744},
journal = {Frontiers in Pharmacology},
number = ,
volume = 10,
place = {Switzerland},
year = {2019},
month = {7}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.3389/fphar.2019.00744

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Cited by: 9 works
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Works referencing / citing this record:

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Nanodisc-solubilized membrane protein library reflects the membrane proteome
journal, February 2013

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Biomimetic tethered lipid membranes designed for membrane-protein interaction studies
journal, July 2007


Establishment and characterization of cell-free translation/glycosylation in insect cell ( Spodoptera frugiperda 21) extract prepared with high pressure treatment
journal, May 2001

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Phosphoinositide-incorporated lipid–protein nanodiscs: A tool for studying protein–lipid interactions
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Assembly of single bacteriorhodopsin trimers in bilayer nanodiscs
journal, June 2006

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Lipid diffusion in planar membranes investigated by fluorescence correlation spectroscopy
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α-Hemolysin pore formation into a supported phospholipid bilayer using cell-free expression
journal, January 2011

  • Chalmeau, Jerome; Monina, Nadezda; Shin, Jonghyeon
  • Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1808, Issue 1
  • DOI: 10.1016/j.bbamem.2010.07.027

Lipid–protein interactions in biological membranes: A dynamic perspective
journal, February 2012


Real-time monitoring of binding events on a thermostabilized human A2A receptor embedded in a lipid bilayer by surface plasmon resonance
journal, May 2015

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  • Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1848, Issue 5
  • DOI: 10.1016/j.bbamem.2015.02.014

Reconstitution of SNARE proteins into solid-supported lipid bilayer stacks and X-ray structure analysis
journal, February 2018

  • Xu, Yihui; Kuhlmann, Jan; Brennich, Martha
  • Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1860, Issue 2
  • DOI: 10.1016/j.bbamem.2017.10.023

Tethered bilayer lipid membranes (tBLMs): Interest and applications for biological membrane investigations
journal, December 2014


Kinetics of lipid mixing between bicelles and nanolipoprotein particles
journal, February 2015


Oriented Membrane Protein Reconstitution into Tethered Lipid Membranes for AFM Force Spectroscopy
journal, November 2016


Epidermal growth factor receptor (EGFR) signaling in cancer
journal, January 2006


The role of HER2 in cancer therapy and targeted drug delivery
journal, September 2010


Cell-free production of G protein-coupled receptors for functional and structural studies
journal, June 2007

  • Klammt, Christian; Schwarz, Daniel; Eifler, Nora
  • Journal of Structural Biology, Vol. 158, Issue 3
  • DOI: 10.1016/j.jsb.2007.01.006

Structural biology of G protein-coupled receptors: new opportunities from XFELs and cryoEM
journal, August 2018

  • Ishchenko, Andrii; Gati, Cornelius; Cherezov, Vadim
  • Current Opinion in Structural Biology, Vol. 51
  • DOI: 10.1016/j.sbi.2018.03.009

Discovery of New GPCR Biology: One Receptor Structure at a Time
journal, January 2009


Stabilizing membrane proteins
journal, August 2001


A novel cell-free translation/glycosylation system prepared from insect cells
journal, January 2000


Preserved Transmembrane Protein Mobility in Polymer-Supported Lipid Bilayers Derived from Cell Membranes
journal, August 2015


Direct Capture of Functional Proteins from Mammalian Plasma Membranes into Nanodiscs
journal, October 2015


Single-Molecule Fluorescence Detection of the Epidermal Growth Factor Receptor in Membrane Discs
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Development of a CHO-Based Cell-Free Platform for Synthesis of Active Monoclonal Antibodies
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Integral Membrane Protein Fragment Recombination after Transfer from Nanolipoprotein Particles to Bicelles
journal, December 2013


Functional and Structural Stability of the Epidermal Growth Factor Receptor in Detergent Micelles and Phospholipid Nanodiscs
journal, September 2008

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  • Biochemistry, Vol. 47, Issue 39
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Modulation of the Cytochrome P450 Reductase Redox Potential by the Phospholipid Bilayer
journal, December 2009

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Design and Synthesis of a Library of BODIPY-Based Environmental Polarity Sensors Utilizing Photoinduced Electron-Transfer-Controlled Fluorescence ON/OFF Switching
journal, May 2007

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Different Apolipoproteins Impact Nanolipoprotein Particle Formation
journal, November 2007

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Hydrogen Production by a Hyperthermophilic Membrane-Bound Hydrogenase in Water-Soluble Nanolipoprotein Particles
journal, June 2009

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Membrane Proteins Solubilized Intact in Lipid Containing Nanoparticles Bounded by Styrene Maleic Acid Copolymer
journal, June 2009

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Structure and Thermodynamics of Lipid Bilayers on Polyethylene Glycol Cushions: Fact and Fiction of PEG Cushioned Membranes
journal, November 2011

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Self-Assembly of Discoidal Phospholipid Bilayer Nanoparticles with Membrane Scaffold Proteins
journal, August 2002

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pH Responsive Polymer Cushions for Probing Membrane Environment Interactions
journal, May 2011

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Insertion of Membrane Proteins into Discoidal Membranes Using a Cell-Free Protein Expression Approach
journal, August 2008

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  • Journal of Proteome Research, Vol. 7, Issue 8
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Structure of the extracellular region of HER2 alone and in complex with the Herceptin Fab
journal, February 2003

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Polymer-supported membranes as models of the cell surface
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Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography
journal, February 2014

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Cryo-EM structure of the ribosome–SecYE complex in the membrane environment
journal, April 2011

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Nanodiscs for structural and functional studies of membrane proteins
journal, June 2016

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Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery
journal, July 2018

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Qualifying a eukaryotic cell-free system for fluorescence based GPCR analyses
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Nanodelivery of a functional membrane receptor to manipulate cellular phenotype
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Fixed-target protein serial microcrystallography with an x-ray free electron laser
journal, August 2014

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Cell-free expression of functional receptor tyrosine kinases
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A new functional membrane protein microarray based on tethered phospholipid bilayers
journal, January 2018

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Express incorporation of membrane proteins from various human cell types into phospholipid bilayer nanodiscs
journal, April 2017

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A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein
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Humanization of an anti-p185HER2 antibody for human cancer therapy.
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Amphipols: Polymers that keep membrane proteins soluble in aqueous solutions
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Identification of a Region within the ErbB2/HER2 Intracellular Domain That Is Necessary for Ligand-independent Association
journal, May 2002

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Transducin Activation by Nanoscale Lipid Bilayers Containing One and Two Rhodopsins
journal, March 2007

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Detergents as Tools in Membrane Biochemistry
journal, June 2001

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Cell-free Co-expression of Functional Membrane Proteins and Apolipoprotein, Forming Soluble Nanolipoprotein Particles
journal, July 2008

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Serial Femtosecond Crystallography of G Protein–Coupled Receptors
journal, May 2018


EGFR Mutations and Lung Cancer
journal, February 2011


Cell-free synthesis of a functional G protein-coupled receptor complexed with nanometer scale bilayer discs
journal, January 2011

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Characterization of De Novo Synthesized GPCRs Supported in Nanolipoprotein Discs
journal, September 2012


Formation and Characterization of Supported Lipid Bilayers Composed of Hydrogenated and Deuterated Escherichia coli Lipids
journal, December 2015


Expression and Association of the Yersinia pestis Translocon Proteins, YopB and YopD, Are Facilitated by Nanolipoprotein Particles
journal, March 2016


Synthesis and biochemical characterization of EGF receptor in a water-soluble membrane model system
journal, June 2017


The power, pitfalls and potential of the nanodisc system for NMR-based studies
journal, December 2016

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Nanodiscs and solution NMR: preparation, application and challenges
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Functional reconstitution of β 2 -adrenergic receptors utilizing self-assembling Nanodisc technology
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Latest Development in Drug Discovery on G Protein-coupled Receptors
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Large-scale production and protein engineering of G protein-coupled receptors for structural studies
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Characterization and Purification of Polydisperse Reconstituted Lipoproteins and Nanolipoprotein Particles
journal, July 2009

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Challenges in the Development of Functional Assays of Membrane Proteins
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Tethered and Polymer Supported Bilayer Lipid Membranes: Structure and Function
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<em>Escherichia coli</em>-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology
journal, January 2019

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Single-particle cryo-EM structure of a voltage-activated potassium channel in lipid nanodiscs
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The ErbB2 Signaling Network as a Target for Breast Cancer Therapy
journal, January 2006

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Atomic force microscopy differentiates discrete size distributions between membrane protein containing and empty nanolipoprotein particles
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Design of glycosylation sites by rapid synthesis and analysis of glycosyltransferases
journal, May 2018

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