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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 Lab. (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. doi: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. doi: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
DOI: 10.3389/fphar.2019.00744

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