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Biochemical Society Transactions Jun 2021In the twelve years since styrene maleic acid (SMA) was first used to extract and purify a membrane protein within a native lipid bilayer, this technological... (Review)
Review
In the twelve years since styrene maleic acid (SMA) was first used to extract and purify a membrane protein within a native lipid bilayer, this technological breakthrough has provided insight into the structural and functional details of protein-lipid interactions. Most recently, advances in cryo-EM have demonstrated that SMA-extracted membrane proteins are a rich-source of structural data. For example, it has been possible to resolve the details of annular lipids and protein-protein interactions within complexes, the nature of lipids within central cavities and binding pockets, regions involved in stabilising multimers, details of terminal residues that would otherwise remain unresolved and the identification of physiologically relevant states. Functionally, SMA extraction has allowed the analysis of membrane proteins that are unstable in detergents, the characterization of an ultrafast component in the kinetics of electron transfer that was not possible in detergent-solubilised samples and quantitative, real-time measurement of binding assays with low concentrations of purified protein. While the use of SMA comes with limitations such as its sensitivity to low pH and divalent cations, its major advantage is maintenance of a protein's lipid bilayer. This has enabled researchers to view and assay proteins in an environment close to their native ones, leading to new structural and mechanistic insights.
Topics: Cryoelectron Microscopy; Lipid Bilayers; Maleates; Membrane Lipids; Membrane Proteins; Polystyrenes; Protein Binding; Protein Conformation; Protein Stability
PubMed: 34110372
DOI: 10.1042/BST20201067 -
Methods in Molecular Biology (Clifton,... 2019Targeted proteomics detects proteins of interest with high sensitivity, quantitative accuracy, and reproducibility. In a targeted proteomics assay, surrogate peptides...
Targeted proteomics detects proteins of interest with high sensitivity, quantitative accuracy, and reproducibility. In a targeted proteomics assay, surrogate peptides are generated by proteolytic digestion of target proteins and selected reaction monitoring (SRM) assays are developed to quantify these peptides using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this report, we describe the details of quantitative analysis of target protein in cells and tissue samples.
Topics: Chromatography, Liquid; Humans; Peptides; Proteins; Proteomics; Reproducibility of Results; Tandem Mass Spectrometry
PubMed: 30276745
DOI: 10.1007/978-1-4939-8814-3_17 -
Current Protocols in Protein Science Feb 2016Selective precipitation of proteins can be used as a bulk method to recover the majority of proteins from a crude lysate, as a selective method to fractionate a subset... (Review)
Review
Selective precipitation of proteins can be used as a bulk method to recover the majority of proteins from a crude lysate, as a selective method to fractionate a subset of proteins from a protein solution, or as a very specific method to recover a single protein of interest from a purification step. This unit describes a number of methods suitable for selective precipitation. In each of the protocols that are outlined, the physical or chemical basis of the precipitation process, the parameters that can be varied for optimization, and the basic steps for developing an optimized precipitation are described.
Topics: Fractional Precipitation; Proteins; Solubility
PubMed: 26836410
DOI: 10.1002/0471140864.ps0405s83 -
Biochimica Et Biophysica Acta. Gene... Feb 2021Gcn5 serves as the defining member of the Gcn5-related N-acetyltransferase (GNAT) superfamily of proteins that display a common structural fold and catalytic mechanism... (Review)
Review
Gcn5 serves as the defining member of the Gcn5-related N-acetyltransferase (GNAT) superfamily of proteins that display a common structural fold and catalytic mechanism involving the transfer of the acyl-group, primarily acetyl-, from CoA to an acceptor nucleophile. In the case of Gcn5, the target is the ε-amino group of lysine primarily on histones. Over the years, studies on Gcn5 structure-function have often formed the basis by which we understand the complex activities and regulation of the entire protein acetyltransferase family. It is now appreciated that protein acetylation occurs on thousands of proteins and can reversibly regulate the function of many cellular processes. In this review, we provide an overview of our fundamental understanding of catalysis, regulation of activity and substrate selection, and inhibitor development for this archetypal acetyltransferase.
Topics: Acetyl Coenzyme A; Acetylation; Biocatalysis; Crystallography; Drug Development; Enzyme Inhibitors; Epigenesis, Genetic; Gene Expression Regulation, Developmental; Histone Acetyltransferases; Histones; Lysine; Models, Molecular; Multienzyme Complexes; Protein Domains; Recombinant Proteins; Saccharomyces cerevisiae Proteins; Structure-Activity Relationship; Substrate Specificity; Transcriptional Activation; p300-CBP Transcription Factors
PubMed: 32841743
DOI: 10.1016/j.bbagrm.2020.194627 -
Methods in Cell Biology 2018The functional form of elastin is a highly cross-linked polymer that organizes as sheets or fibers in the extracellular matrix. Purification of the mature protein is...
The functional form of elastin is a highly cross-linked polymer that organizes as sheets or fibers in the extracellular matrix. Purification of the mature protein is problematic because its insolubility precludes its isolation using standard wet-chemistry techniques. Instead, relatively harsh experimental approaches designed to remove nonelastin "contaminates" are employed to generate an insoluble product that has the amino acid composition expected of elastin. Although soluble, tropoelastin also presents problems for isolation and purification. The protein's extreme stickiness and susceptibility to proteolysis require careful attention during purification and in tropoelastin-based assays. This chapter describes the most common approaches for purification of elastin and for preparing solubilized forms of the protein.
Topics: Alkalies; Animals; Elastin; Extracellular Matrix; Hot Temperature; Microscopy, Electron; Molecular Imaging; Proteolysis; Solubility; Staining and Labeling
PubMed: 29310779
DOI: 10.1016/bs.mcb.2017.08.012 -
Biotechnology Advances 2017The current chromatographic approaches used in protein purification are not keeping pace with the increasing biopharmaceutical market demand. With the upstream... (Review)
Review
The current chromatographic approaches used in protein purification are not keeping pace with the increasing biopharmaceutical market demand. With the upstream improvements, the bottleneck shifted towards the downstream process. New approaches rely in Anything But Chromatography methodologies and revisiting former techniques with a bioprocess perspective. Protein crystallization and precipitation methods are already implemented in the downstream process of diverse therapeutic biological macromolecules, overcoming the current chromatographic bottlenecks. Promising work is being developed in order to implement crystallization and precipitation in the purification pipeline of high value therapeutic molecules. This review focuses in the role of these two methodologies in current industrial purification processes, and highlights their potential implementation in the purification pipeline of high value therapeutic molecules, overcoming chromatographic holdups.
Topics: Biopharmaceutics; Chemical Precipitation; Crystallization; Proteins
PubMed: 27908674
DOI: 10.1016/j.biotechadv.2016.11.005 -
Bioscience Reports Jul 2021Protein purification is the vital basis to study the function, structure and interaction of proteins. Widely used methods are affinity chromatography-based...
Protein purification is the vital basis to study the function, structure and interaction of proteins. Widely used methods are affinity chromatography-based purifications, which require different chromatography columns and harsh conditions, such as acidic pH and/or adding imidazole or high salt concentration, to elute and collect the purified proteins. Here we established an easy and fast purification method for soluble proteins under mild conditions, based on the light-induced protein dimerization system improved light-induced dimer (iLID), which regulates protein binding and release with light. We utilize the biological membrane, which can be easily separated by centrifugation, as the port to anchor the target proteins. In Xenopus laevis oocyte and Escherichia coli, the blue light-sensitive part of iLID, AsLOV2-SsrA, was targeted to the plasma membrane by different membrane anchors. The other part of iLID, SspB, was fused with the protein of interest (POI) and expressed in the cytosol. The SspB-POI can be captured to the membrane fraction through light-induced binding to AsLOV2-SsrA and then released purely to fresh buffer in the dark after simple centrifugation and washing. This method, named mem-iLID, is very flexible in scale and economic. We demonstrate the quickly obtained yield of two pure and fully functional enzymes: a DNA polymerase and a light-activated adenylyl cyclase. Furthermore, we also designed a new SspB mutant for better dissociation and less interference with the POI, which could potentially facilitate other optogenetic manipulations of protein-protein interaction.
Topics: Adenylyl Cyclases; Animals; Cell Membrane; Cost-Benefit Analysis; DNA-Directed DNA Polymerase; Escherichia coli; Escherichia coli Proteins; Light; Mutation; Optogenetics; Protein Binding; Protein Engineering; Protein Multimerization; Recombinant Fusion Proteins; Time Factors; Workflow; Xenopus Proteins; Xenopus laevis
PubMed: 34142112
DOI: 10.1042/BSR20210800 -
Methods in Molecular Biology (Clifton,... 2017Separation science continues to occupy the central position in the overall strategy for the downstream processing and purification of therapeutic protein products for... (Review)
Review
Separation science continues to occupy the central position in the overall strategy for the downstream processing and purification of therapeutic protein products for human use. Increasing product titers from mammalian cell culture and new emerging classes of biopharmaceuticals has presented a challenge to the industry to identify ways of improving the robustness and economics of chromatography processes. In commercial manufacturing, there is always a need to increase the scale of the chromatography operations which are typically developed and optimized in small-scale laboratory experiments. This review discusses the key factors in the chromatography process that need to be considered as the scale of the purification step is increased in order to maintain the purity and integrity of the product purified at smaller scale.
Topics: Biotechnology; Chromatography; Proteins
PubMed: 27730549
DOI: 10.1007/978-1-4939-6412-3_5 -
Methods in Molecular Biology (Clifton,... 2020Biochemical, biophysical, and structural studies of membrane proteins rely on the availability of highly pure and monodisperse membrane protein samples. One of the most...
Biochemical, biophysical, and structural studies of membrane proteins rely on the availability of highly pure and monodisperse membrane protein samples. One of the most powerful methods for isolation of the membrane protein of interest is affinity purification. This methodology typically relies on engineering an affinity tag into the protein of interest and an affinity resin that specifically recognizes the tag, allowing one to purify the target protein in a single step. In some cases, the affinity purification procedure is combined with additional steps to increase the purity and homogeneity of the final protein sample. Here, we describe several protocols for affinity purification of TSPO, a small membrane protein. The techniques we use include immobilized metal affinity chromatography (IMAC) and strep-II tag-based streptavidin affinity chromatography.
Topics: Affinity Labels; Amino Acid Sequence; Animals; Chromatography, Affinity; Detergents; Escherichia coli; Eukaryotic Cells; Histidine; Humans; Insecta; Ion Exchange Resins; Membrane Proteins; Oligopeptides; Recombinant Fusion Proteins; Recombinant Proteins; Solubility; Streptavidin
PubMed: 32112319
DOI: 10.1007/978-1-0716-0373-4_9 -
Methods in Molecular Biology (Clifton,... 2021Membrane proteins constitute an important class of proteins for medical, pharmaceutical, and biotechnological reasons. Understanding the structure and function of...
Membrane proteins constitute an important class of proteins for medical, pharmaceutical, and biotechnological reasons. Understanding the structure and function of membrane proteins and their complexes is of key importance, but the progress in this area is slow because of the difficulties to produce them in sufficient quality and quantity. Overexpression of membrane proteins is often restricted by the limited capability of translocation systems to integrate proteins into the membrane and to fold them properly. Purification of membrane proteins requires their isolation from the membrane, which is a further challenge. The choice of expression system, detergents, and purification tags is therefore an important decision. Here, we present a protocol for expression in bacteria and isolation of a seven-subunit membrane protein complex, the bacterial holo-translocon, which can serve as a starting point for the production of other membrane protein complexes for structural and functional studies.
Topics: Chromatography, Affinity; Chromatography, Gel; Escherichia coli; Gene Expression; Membrane Proteins; Multiprotein Complexes; Plasmids; Promoter Regions, Genetic; Protein Multimerization; Protein Subunits; Recombinant Proteins
PubMed: 33301109
DOI: 10.1007/978-1-0716-1126-5_1