-
Cellular and Molecular Life Sciences :... May 2006G protein-coupled receptors (GPCRS) represent a class of integral membrane proteins involved in many biological processes and pathologies. Fifty percent of all modern... (Comparative Study)
Comparative Study Review
G protein-coupled receptors (GPCRS) represent a class of integral membrane proteins involved in many biological processes and pathologies. Fifty percent of all modern drugs and almost 25% of the top 200 bestselling drugs are estimated to target GPCRs. Despite these crucial biological implications, very little is known, at atomic resolution, about the detailed molecular mechanisms by which these membrane proteins are able to recognize their extra-cellular stimuli and transmit the associated messages. Obviously, our understanding of GPCR functioning would be greatly facilitated by the availability of high-resolution three-dimensional (3D) structural data. However, expression, solubilization and purification of these membrane proteins are not easy to achieve, and at present, only one 3D structure has been determined, that of bovine rhodopsin. This review presents and compares the different successful strategies which have been applied to solubilize and purify recombinant GPCRs in the perspective of structural biology experiments.
Topics: Animals; Chromatography; Detergents; Humans; Protein Renaturation; Receptors, G-Protein-Coupled; Recombinant Proteins; Solubility
PubMed: 16568239
DOI: 10.1007/s00018-005-5557-6 -
Microbial Cell Factories Jun 2016Formation of inclusion bodies poses a major hurdle in recovery of bioactive recombinant protein from Escherichia coli. Urea and guanidine hydrochloride have routinely...
BACKGROUND
Formation of inclusion bodies poses a major hurdle in recovery of bioactive recombinant protein from Escherichia coli. Urea and guanidine hydrochloride have routinely been used to solubilize inclusion body proteins, but many times result in poor recovery of bioactive protein. High pH buffers, detergents and organic solvents like n-propanol have been successfully used as mild solubilization agents for high throughput recovery of bioactive protein from bacterial inclusion bodies. These mild solubilization agents preserve native-like secondary structures of proteins in inclusion body aggregates and result in improved recovery of bioactive protein as compared to conventional solubilization agents. Here we demonstrate solubilization of human growth hormone inclusion body aggregates using 30% trifluoroethanol in presence of 3 M urea and its refolding into bioactive form.
RESULTS
Human growth hormone was expressed in E. coli M15 (pREP) cells in the form of inclusion bodies. Different concentrations of trifluoroethanol with or without addition of low concentration (3 M) of urea were used for solubilization of inclusion body aggregates. Thirty percent trifluoroethanol in combination with 3 M urea was found to be suitable for efficient solubilization of human growth hormone inclusion bodies. Solubilized protein was refolded by dilution and purified by anion exchange and size exclusion chromatography. Purified protein was analyzed for secondary and tertiary structure using different spectroscopic tools and was found to be bioactive by cell proliferation assay. To understand the mechanism of action of trifluoroethanol, secondary and tertiary structure of human growth hormone in trifluoroethanol was compared to that in presence of other denaturants like urea and guanidine hydrochloride. Trifluoroethanol was found to be stabilizing the secondary structure and destabilizing the tertiary structure of protein. Finally, it was observed that trifluoroethanol can be used to solubilize inclusion bodies of a number of proteins.
CONCLUSIONS
Trifluoroethanol was found to be a suitable mild solubilization agent for bacterial inclusion bodies. Fully functional, bioactive human growth hormone was recovered in high yield from inclusion bodies using trifluoroethanol based solubilization buffer. It was also observed that trifluoroethanol has potential to solubilize inclusion bodies of different proteins.
Topics: Chromatography, Gel; Chromatography, High Pressure Liquid; Circular Dichroism; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Human Growth Hormone; Humans; Inclusion Bodies; Protein Refolding; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Proteins; Trifluoroethanol
PubMed: 27277580
DOI: 10.1186/s12934-016-0504-9 -
Acta Biochimica Et Biophysica Sinica Dec 2009Overexpression of foreign proteins in Escherichia coli often leads to the formation of inclusion bodies (IBs), which becomes the major bottleneck in the preparation of...
Overexpression of foreign proteins in Escherichia coli often leads to the formation of inclusion bodies (IBs), which becomes the major bottleneck in the preparation of recombinant proteins and their applications. In the present study, 36 proteins from IBs were refolded using a simple refolding method. Refolding yields of these proteins were defined as the percentage of soluble proteins following dilution refolding in the amount of denatured proteins in the samples before diluting into refolding buffer. Furthermore, a mathematical model was deduced to evaluate the role of biochemical properties in the protein refolding. Our results indicated that under the experimental conditions, isoelectric point of proteins might be mostly contributing to the high efficacy of protein refolding since the increment of one unit resulted in a decrease of 14.83% in the refolding yield. Other important mediators were components of protein secondary structure and the molecular weight (R(2) = 0.98, P = 0.000, F-test). Six proteins with low efficiency in the protein refolding possessed relatively low isoelectric points. Furthermore, refolding yields of six additional proteins from IBs were predicted and further validated by refolding the proteins under the same conditions. Therefore, the model of protein refolding developed here could be used to predict the refolding yields of proteins from IBs through a simple method. Our study will be suggestive to optimize the methods for protein refolding from IBs according to their intrinsic properties.
Topics: Buffers; Escherichia coli; Escherichia coli Proteins; Gene Expression; Inclusion Bodies; Isoelectric Point; Models, Biological; Molecular Weight; Protein Folding; Protein Structure, Secondary; Solubility
PubMed: 20011979
DOI: 10.1093/abbs/gmp098 -
Molecules (Basel, Switzerland) Feb 2017Conformational changes of d-glucose/d-galactose-binding protein (GGBP) were studied under molecular crowding conditions modeled by concentrated solutions of polyethylene...
Conformational changes of d-glucose/d-galactose-binding protein (GGBP) were studied under molecular crowding conditions modeled by concentrated solutions of polyethylene glycols (PEG-12000, PEG-4000, and PEG-600), Ficoll-70, and Dextran-70, addition of which induced noticeable structural changes in the GGBP molecule. All PEGs promoted compaction of GGBP and lead to the increase in ordering of its structure. Concentrated solutions of PEG-12000 and PEG-4000 caused GGBP aggregation. Although Ficoll-70 and Dextran-70 also promoted increase in the GGBP ordering, the structural outputs were different for different crowders. For example, in comparison with the GGBP in buffer, the intrinsic fluorescence spectrum of this protein was shifted to short-wave region in the presence of PEGs but was red-shifted in the presence of Ficoll-70 and Dextran-70. It was hypothesized that this difference could be due to the specific interaction of GGBP with the sugar-based polymers (Ficoll-70 and Dextran-70), indicating that protein can adopt different conformations in solutions containing molecular crowders of different chemical nature. It was also shown that all tested crowding agents were able to stabilize GGBP structure shifting the GGBP guanidine hydrochloride (GdnHCl)-induced unfolding curves to higher denaturant concentrations, but their stabilization capabilities did not depend on the hydrodynamic dimensions of the polymers molecules. Refolding of GGBP was complicated by protein aggregation in all tested solutions of crowding agents. The lowest yield of refolded protein was achieved in the highly concentrated solutions of PEG-12000. These data support the previous notion that the influence of macromolecular crowders on proteins is rather complex phenomenon that extends beyond the excluded volume effects.
Topics: Calcium-Binding Proteins; Glucose; Models, Molecular; Molecular Conformation; Monosaccharide Transport Proteins; Periplasmic Binding Proteins; Protein Refolding; Protein Unfolding; Structure-Activity Relationship
PubMed: 28178192
DOI: 10.3390/molecules22020244 -
Scientific Reports May 2016Production of membrane-associated cell surface receptors and their ligands is often a cumbersome, expensive, and time-consuming process that limits detailed structural...
Production of membrane-associated cell surface receptors and their ligands is often a cumbersome, expensive, and time-consuming process that limits detailed structural and functional characterization of this important class of proteins. Here we report a rapid method for refolding inclusion-body-based, recombinant cell surface receptors and ligands in one day, a speed equivalent to that of soluble protein production. This method efficiently couples modular on-column immobilized metal ion affinity purification and solid-phase protein refolding. We demonstrated the general utility of this method for producing multiple functionally active immunoreceptors, ligands, and viral decoys, including challenging cell surface proteins that cannot be produced using typical dialysis- or dilution-based refolding approaches.
Topics: Circular Dichroism; Inclusion Bodies; Ligands; Models, Molecular; Protein Refolding; Receptors, Cell Surface; Recombinant Proteins
PubMed: 27215173
DOI: 10.1038/srep26482 -
Nature Feb 2021A non-enveloped virus requires a membrane lesion to deliver its genome into a target cell. For rotaviruses, membrane perforation is a principal function of the viral...
A non-enveloped virus requires a membrane lesion to deliver its genome into a target cell. For rotaviruses, membrane perforation is a principal function of the viral outer-layer protein, VP4. Here we describe the use of electron cryomicroscopy to determine how VP4 performs this function and show that when activated by cleavage to VP8* and VP5*, VP4 can rearrange on the virion surface from an 'upright' to a 'reversed' conformation. The reversed structure projects a previously buried 'foot' domain outwards into the membrane of the host cell to which the virion has attached. Electron cryotomograms of virus particles entering cells are consistent with this picture. Using a disulfide mutant of VP4, we have also stabilized a probable intermediate in the transition between the two conformations. Our results define molecular mechanisms for the first steps of the penetration of rotaviruses into the membranes of target cells and suggest similarities with mechanisms postulated for other viruses.
Topics: Animals; Antigens, Viral; Capsid Proteins; Cell Line; Cell Membrane; Cryoelectron Microscopy; Disulfides; Models, Molecular; Mutant Proteins; Mutation; Protein Conformation; Protein Refolding; RNA-Binding Proteins; Rotavirus; Viral Nonstructural Proteins; Virion; Virus Internalization
PubMed: 33442061
DOI: 10.1038/s41586-020-03124-4 -
Journal of Visualized Experiments : JoVE Dec 2015The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the...
The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His)6-tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni(2+)-charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner.
Topics: Amino Acid Sequence; Amyloid; Animals; Chromatography, Gel; Escherichia coli; GPI-Linked Proteins; Histidine; Inclusion Bodies; Mice; Molecular Sequence Data; Nerve Tissue Proteins; Oligopeptides; Protein Folding; Protein Renaturation; Recombinant Proteins; Urea
PubMed: 26709825
DOI: 10.3791/53432 -
Biochimica Et Biophysica Acta.... Feb 2020Sequestration of misfolded proteins into distinct cellular compartments plays a pivotal role in proteostasis and proteopathies. Cytoplasmic ubiquitinated proteins are...
Sequestration of misfolded proteins into distinct cellular compartments plays a pivotal role in proteostasis and proteopathies. Cytoplasmic ubiquitinated proteins are sequestered by p62/SQSTM1 to deposit in sequestosomes or aggresome-like induced structures (ALIS). Most aggresome or ALIS regulators identified thus far are recruiters, while little is known about the disaggregases or dissolvers. In this research, we showed that lanosterol synthase and its enzymatic product lanosterol effectively reduced the number and/or size of sequestosomes/ALIS/aggresomes formed by endogenous proteins in the HeLa and HEK-293A cells cultured under both non-stressed and stressed conditions. Supplemented lanosterol did not affect the proteasome and autophagic activities, but released the trapped proteins from the p62-positive inclusions accompanied with the activation of HSF1 and up-regulation of various heat shock proteins. Our results suggested that the coordinated actions of disaggregation by lanosterol and refolding by heat shock proteins might facilitate the cells to recycle proteins from aggregates. The disaggregation activity of lanosterol was not shared by cholesterol, indicating that lanosterol possesses additional cellular functions in proteostasis regulation. Our findings highlight that besides protein modulators, the cells also possess endogenous low-molecular-weight compounds as efficient proteostasis regulators.
Topics: Cell Survival; Cytosol; HEK293 Cells; HeLa Cells; Heat Shock Transcription Factors; Heat-Shock Proteins; Humans; Lanosterol; Leupeptins; Protein Aggregates; Protein Refolding; Proteostasis; Sequestosome-1 Protein; Temperature; Up-Regulation
PubMed: 31785334
DOI: 10.1016/j.bbamcr.2019.118617 -
ELife Feb 2014The small molecule EMD 57033 has been shown to stimulate the actomyosin ATPase activity and contractility of myofilaments. Here, we show that EMD 57033 binds to an...
The small molecule EMD 57033 has been shown to stimulate the actomyosin ATPase activity and contractility of myofilaments. Here, we show that EMD 57033 binds to an allosteric pocket in the myosin motor domain. EMD 57033-binding protects myosin against heat stress and thermal denaturation. In the presence of EMD 57033, ATP hydrolysis, coupling between actin and nucleotide binding sites, and actin affinity in the presence of ATP are increased more than 10-fold. Addition of EMD 57033 to heat-inactivated β-cardiac myosin is followed by refolding and reactivation of ATPase and motile activities. In heat-stressed cardiomyocytes expression of the stress-marker atrial natriuretic peptide is suppressed by EMD 57033. Thus, EMD 57033 displays a much wider spectrum of activities than those previously associated with small, drug-like compounds. Allosteric effectors that mediate refolding and enhance enzymatic function have the potential to improve the treatment of heart failure, myopathies, and protein misfolding diseases. DOI: http://dx.doi.org/10.7554/eLife.01603.001.
Topics: Actins; Adenosine Triphosphate; Allosteric Regulation; Animals; Animals, Newborn; Binding Sites; Cardiac Myosins; Cardiotonic Agents; Catalytic Domain; Cells, Cultured; Dictyostelium; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Activators; Humans; Hydrolysis; Kinetics; Molecular Docking Simulation; Myocardial Contraction; Myocytes, Cardiac; Protein Conformation; Protein Refolding; Quinolines; Rats; Recombinant Proteins; Structure-Activity Relationship; Thiadiazines
PubMed: 24520162
DOI: 10.7554/eLife.01603 -
Nature Protocols 2014An understanding of the mechanism accompanying functional conformational changes associated with protein activation has important implications for drug design. Here we...
An understanding of the mechanism accompanying functional conformational changes associated with protein activation has important implications for drug design. Here we describe a powerful method, conformational changes and dynamics using stable-isotope labeling and mass spectrometry (CDSiL-MS), which involves chemical labeling by isotope-coded forms of N-ethylmaleimide or succinic anhydride to site-specifically label the side chains of cysteines or lysines, respectively, in native proteins. Subsequent MS analysis allows the quantitative monitoring of reactivity of residues as a function of time, providing a measurement of the labeling kinetics and thereby enabling elucidation of conformational changes of proteins. We demonstrate the utility of this method using a model G protein-coupled receptor, the β2-adrenergic receptor, including experiments that characterize the functional conformational changes associated with activation of distinct signaling pathways induced by different β-adrenoceptor ligands. The procedure requires 5 d, and it can easily be adapted to systems in which soluble and detergent-solubilized membrane protein targets, which undergo function-dependent conformational changes, can be interrogated structurally to allow drug screening.
Topics: Ethylmaleimide; Isotope Labeling; Kinetics; Mass Spectrometry; Protein Conformation; Protein Refolding; Proteins; Receptors, Adrenergic, beta-2; Succinic Anhydrides
PubMed: 24810039
DOI: 10.1038/nprot.2014.075