Browse
Submit Data
Databases
API
Help

Dataset Information

8 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Expression and purification of recombinant G protein-coupled receptors: A review.

ABSTRACT: Given their extensive role in cell signalling, GPCRs are significant drug targets; despite this, many of these receptors have limited or no available prophylaxis. Novel drug design and discovery significantly rely on structure determination, of which GPCRs are typically elusive. Progress has been made thus far to produce sufficient quantity and quality of protein for downstream analysis. As such, this review highlights the systems available for recombinant GPCR expression, with consideration of their advantages and disadvantages, as well as examples of receptors successfully expressed in these systems. Additionally, an overview is given on the use of detergents and the styrene maleic acid (SMA) co-polymer for membrane solubilisation, as well as purification techniques.

SUBMITTER: Wiseman DN

PROVIDER: S-EPMC6983937 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Expression and purification of recombinant G protein-coupled receptors: A review.

Wiseman Daniel N DN Otchere Abigail A Patel Jaimin H JH Uddin Romez R Pollock Naomi L NL Routledge Sarah J SJ Rothnie Alice J AJ Slack Cathy C Poyner David R DR Bill Roslyn M RM Goddard Alan D AD

Protein expression and purification 20191031

Given their extensive role in cell signalling, GPCRs are significant drug targets; despite this, many of these receptors have limited or no available prophylaxis. Novel drug design and discovery significantly rely on structure determination, of which GPCRs are typically elusive. Progress has been made thus far to produce sufficient quantity and quality of protein for downstream analysis. As such, this review highlights the systems available for recombinant GPCR expression, with consideration of ...[more]

PMID: 31678667

Similar Datasets

Expression analysis of G Protein-Coupled Receptors in mouse macrophages.

Project description:BACKGROUND:Monocytes and macrophages express an extensive repertoire of G Protein-Coupled Receptors (GPCRs) that regulate inflammation and immunity. In this study we performed a systematic micro-array analysis of GPCR expression in primary mouse macrophages to identify family members that are either enriched in macrophages compared to a panel of other cell types, or are regulated by an inflammatory stimulus, the bacterial product lipopolysaccharide (LPS). RESULTS:Several members of the P2RY family had striking expression patterns in macrophages; P2ry6 mRNA was essentially expressed in a macrophage-specific fashion, whilst P2ry1 and P2ry5 mRNA levels were strongly down-regulated by LPS. Expression of several other GPCRs was either restricted to macrophages (e.g. Gpr84) or to both macrophages and neural tissues (e.g. P2ry12, Gpr85). The GPCR repertoire expressed by bone marrow-derived macrophages and thioglycollate-elicited peritoneal macrophages had some commonality, but there were also several GPCRs preferentially expressed by either cell population. CONCLUSION:The constitutive or regulated expression in macrophages of several GPCRs identified in this study has not previously been described. Future studies on such GPCRs and their agonists are likely to provide important insights into macrophage biology, as well as novel inflammatory pathways that could be future targets for drug discovery.

| S-EPMC2394514 | biostudies-literature

G Protein-Coupled Receptors

Project description: Not available

| S-EPMC3315628 | biostudies-literature

Modular detergents tailor the purification and structural analysis of membrane proteins including G-protein coupled receptors.

Project description:Detergents enable the purification of membrane proteins and are indispensable reagents in structural biology. Even though a large variety of detergents have been developed in the last century, the challenge remains to identify guidelines that allow fine-tuning of detergents for individual applications in membrane protein research. Addressing this challenge, here we introduce the family of oligoglycerol detergents (OGDs). Native mass spectrometry (MS) reveals that the modular OGD architecture offers the ability to control protein purification and to preserve interactions with native membrane lipids during purification. In addition to a broad range of bacterial membrane proteins, OGDs also enable the purification and analysis of a functional G-protein coupled receptor (GPCR). Moreover, given the modular design of these detergents, we anticipate fine-tuning of their properties for specific applications in structural biology. Seen from a broader perspective, this represents a significant advance for the investigation of membrane proteins and their interactions with lipids.

| S-EPMC6987200 | biostudies-literature

Expression and purification of recombinant protein related to DAN and cerberus (PRDC).

Project description:Bone morphogenetic proteins (BMPs) are secreted protein ligands that control numerous biological processes, such as cell differentiation and cell proliferation. Ligands are regulated by a large number of structurally diverse extracellular antagonists. PRDC or protein related to DAN and cerberus is a BMP antagonist of the DAN family, which is defined by a conserved pattern of cysteine residues that form a ring structure. Here we present the expression and purification of recombinant mouse PRDC (mPRDC) from bacterial (Escherichia coli) inclusion bodies through oxidative refolding. Functional mPRDC was isolated from a nonfunctional component through reverse phase chromatography and shown to inhibit BMP2 and BMP4 in a cell-based luciferase reporter assay. Recombinant mPRDC also bound directly to BMP2, BMP4 and BMP7, but not activin A. Furthermore, circular dichroism indicated that mPRDC is folded and contains a higher than anticipated helical content for a DAN family member protein.

| S-EPMC3319168 | biostudies-literature

Expression and purification of recombinant Saccharomyces cerevisiae mitochondrial carrier protein YGR257Cp (Mtm1p).

Project description:The Saccharomyces cerevisiae mitochondrial carrier YGR257Cp (Mtm1p) is an integral membrane protein that plays an essential role in mitochondrial iron homeostasis and respiratory functions, but its carrier substrate has not previously been identified. Large amounts of pure protein are required for biochemical characterization, including substrate screening. Functional complementation of a Saccharomyces knockout by expression of TwinStrep tagged YGR257Cp demonstrates that an affinity tag does not interfere with protein function, but the expression level is very low. Heterologous expression in Pichia pastoris improves the yield but the product is heterogeneous. Expression has been screened in several Escherichia coli hosts, optimizing yield by modifying induction conditions and supplementing with rare tRNAs to overcome codon bias in the eukaryotic gene. Detection of an additional N-terminal truncation product in E. coli reveals the presence of a secondary intracistronic translation initiation site, which can be eliminated by silent mutagenesis of an alternative (Leu) initiation codon, resulting in production of a single, full-length polypeptide (∼30% of the total protein) as insoluble inclusion bodies. Purified inclusion bodies were successfully refolded and affinity purified, yielding approximately 40mg of pure, soluble product per liter of culture. Refolded YGR257Cp binds pyridoxal 5'-phosphate tightly (KD<1μM), supporting a new hypothesis that the mitochondrial carrier YGR237Cp and its homologs function as high affinity PLP transporters in mitochondria, providing the first evidence for this essential transport function in eukaryotes.

| S-EPMC3876937 | biostudies-literature

The pCri System: a vector collection for recombinant protein expression and purification.

Project description:A major bottleneck in structural, biochemical and biophysical studies of proteins is the need for large amounts of pure homogenous material, which is generally obtained by recombinant overexpression. Here we introduce a vector collection, the pCri System, for cytoplasmic and periplasmic/extracellular expression of heterologous proteins that allows the simultaneous assessment of prokaryotic and eukaryotic host cells (Escherichia coli, Bacillus subtilis, and Pichia pastoris). By using a single polymerase chain reaction product, genes of interest can be directionally cloned in all vectors within four different rare restriction sites at the 5'end and multiple cloning sites at the 3'end. In this way, a number of different fusion tags but also signal peptides can be incorporated at the N- and C-terminus of proteins, facilitating their expression, solubility and subsequent detection and purification. Fusion tags can be efficiently removed by treatment with site-specific peptidases, such as tobacco etch virus proteinase, thrombin, or sentrin specific peptidase 1, which leave only a few extra residues at the N-terminus of the protein. The combination of different expression systems in concert with the cloning approach in vectors that can fuse various tags makes the pCri System a valuable tool for high throughput studies.

| S-EPMC4227841 | biostudies-literature

Cloning, expression and purification of Chikungunya virus E2 recombinant protein in E.coli

Project description: Not available

| S-EPMC4080392 | biostudies-literature

Heterologous expression of functional G-protein-coupled receptors in Caenorhabditis elegans.

Project description:New strategies for expression, purification, functional characterization, and structural determination of membrane-spanning G-protein-coupled receptors (GPCRs) are constantly being developed because of their importance to human health. Here, we report a Caenorhabditis elegans heterologous expression system able to produce milligram amounts of functional native and engineered GPCRs. Both bovine opsin [(b)opsin] and human adenosine A(2A) subtype receptor [(h)A(2A)R] expressed in neurons or muscles of C. elegans were localized to cell membranes. Worms expressing these GPCRs manifested changes in motor behavior in response to light and ligands, respectively. With a newly devised protocol, 0.6-1 mg of purified homogenous 9-cis-retinal-bound bovine isorhodopsin [(b)isoRho] and ligand-bound (h)A(2A)R were obtained from C. elegans from one 10-L fermentation at low cost. Purified recombinant (b)isoRho exhibited its signature absorbance spectrum and activated its cognate G-protein transducin in vitro at a rate similar to native rhodopsin (Rho) obtained from bovine retina. Generally high expression levels of 11 native and mutant GPCRs demonstrated the potential of this C. elegans system to produce milligram quantities of high-quality GPCRs and possibly other membrane proteins suitable for detailed characterization.

| S-EPMC3290431 | biostudies-literature

Multi-Compartment and Multi-Host Vector Suite for Recombinant Protein Expression and Purification.

Project description:Recombinant protein expression has become an invaluable tool in basic and applied research. The accumulated knowledge in this field allowed the expression of thousands of protein targets in a soluble, pure, and homogeneous state, essential for biochemical and structural analyses. A lot of progress has been achieved in the last decades, where challenging proteins were expressed in a soluble manner after evaluating different parameters such as host, strain, and fusion partner or promoter strength, among others. In this regard, we have previously developed a vector suite that allows the evaluation of different promoters and solubility enhancer-proteins, through an easy and efficient cloning strategy. Nonetheless, the proper expression of many targets remains elusive, requiring, for example, the addition of complex post-translation modifications and/or passage through specialized compartments. In order to overcome the limitations found when working with a single subcellular localization and a single host type, we herein expanded our previously developed vector suite to include the evaluation of recombinant protein expression in different cell compartments and cell hosts. In addition, these vectors also allow the assessment of alternative purification strategies for the improvement of target protein yields.

| S-EPMC6030378 | biostudies-literature

Powdered G-Protein-Coupled Receptors.

Project description:Preparation and storage of functional membrane proteins such as G-protein-coupled receptors (GPCRs) are crucial to the processes of drug delivery and discovery. Here, we describe a method of preparing powdered GPCRs using rhodopsin as the prototype. We purified rhodopsin in CHAPS detergent with low detergent to protein ratio so the bulk of the sample represented protein (ca. 72% w/w). Our new method for generating powders of membrane proteins followed by rehydration paves the way for conducting functional and biophysical experiments. As an illustrative application, powdered rhodopsin was prepared with and without the cofactor 11-cis-retinal to enable partial rehydration of the protein with D2O in a controlled manner. Quasi-elastic neutron scattering studies using both spatial motion and energy landscape models form the basis for crucial insights into structural fluctuations and thermodynamics of GPCR activation.

| S-EPMC5386826 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data