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Current Protocols in Protein Science Apr 2015When the first version of this unit was written in 1995, protein purification of recombinant proteins was based on a variety of standard chromatographic methods and... (Review)
Review
When the first version of this unit was written in 1995, protein purification of recombinant proteins was based on a variety of standard chromatographic methods and approaches, many of which were described and mentioned throughout Current Protocols in Protein Science. In the interim, there has been a shift toward an almost universal usage of the affinity or fusion tag. This may not be the case for biotechnology manufacture where affinity tags can complicate producing proteins under regulatory conditions. Regardless of the protein expression system, questions are asked as to which and how many affinity tags to use, where to attach them in the protein, and whether to engineer a self-cleavage system or simply leave them on. We will briefly address some of these issues. Also, although this overview focuses on E.coli, protein expression and purification, other commonly used expression systems are mentioned and, apart from cell-breakage methods, protein purification methods and strategies are essentially the same.
Topics: Affinity Labels; Chromatography, Affinity; Escherichia coli; Recombinant Proteins
PubMed: 25829302
DOI: 10.1002/0471140864.ps0601s80 -
Journal of Molecular Biology Nov 2018The formation of membrane-less organelles and compartments by protein phase separation is an important way in which cells organize their cytoplasm and nucleoplasm. In...
The formation of membrane-less organelles and compartments by protein phase separation is an important way in which cells organize their cytoplasm and nucleoplasm. In vitro phase separation assays with purified proteins have become the standard way to investigate proteins that form membrane-less compartments. By now, various proteins have been purified and tested for their ability to phase separate and form liquid condensates in vitro. However, phase-separating proteins are often aggregation-prone and difficult to purify and handle. As a consequence, the results from phase separation assays often differ between labs and are not easily reproduced. Thus, there is an urgent need for high-quality proteins, standardized procedures, and generally agreed-upon practices for protein purification and conducting phase separation assays. This paper provides protocols for protein purification and guides the user through the practicalities of in vitro protein phase separation assays, including best-practice approaches and pitfalls to avoid. We believe that this compendium of protocols and practices will provide a useful resource for scientists studying the phase behavior of proteins.
Topics: Animals; Cell Nucleus; Chemical Fractionation; Cytoplasm; Guidelines as Topic; In Vitro Techniques; Liquid-Liquid Extraction; Peptide Termination Factors; Phase Transition; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Solid Phase Extraction
PubMed: 29944854
DOI: 10.1016/j.jmb.2018.06.038 -
Methods in Molecular Biology (Clifton,... 2015Western blotting is an important procedure for the immunodetection of proteins, particularly proteins that are of low abundance. This process involves the transfer of... (Review)
Review
Western blotting is an important procedure for the immunodetection of proteins, particularly proteins that are of low abundance. This process involves the transfer of protein patterns from gel to microporous membrane. Electrophoretic as well as non-electrophoretic transfer of proteins to membranes was first described in 1979. Protein blotting has evolved greatly since the inception of this protocol, allowing protein transfer to be accomplished in a variety of ways.
Topics: Blotting, Western; Buffers; Immobilized Proteins; Membranes, Artificial
PubMed: 26043986
DOI: 10.1007/978-1-4939-2694-7_5 -
Methods in Molecular Biology (Clifton,... 2015The binding between biotin and streptavidin or avidin is one of the strongest known non-covalent biological interactions. The (strept)avidin-biotin interaction has been...
The binding between biotin and streptavidin or avidin is one of the strongest known non-covalent biological interactions. The (strept)avidin-biotin interaction has been widely used for decades in biological research and biotechnology. Therefore labeling of purified proteins by biotin is a powerful way to achieve protein capture, immobilization, and functionalization, as well as multimerizing or bridging molecules. Chemical biotinylation often generates heterogeneous products, which may have impaired function. Enzymatic biotinylation with E. coli biotin ligase (BirA) is highly specific in covalently attaching biotin to the 15 amino acid AviTag peptide, giving a homogeneous product with high yield. AviTag can conveniently be added genetically at the N-terminus, C-terminus, or in exposed loops of a target protein. We describe here procedures for AviTag insertion by inverse PCR, purification of BirA fused to glutathione-S-transferase (GST-BirA) from E. coli, BirA biotinylation of purified protein, and gel-shift analysis by SDS-PAGE to quantify the extent of biotinylation.
Topics: Biotin; Biotinylation; Carbon-Nitrogen Ligases; Chromatography, Affinity; Escherichia coli; Escherichia coli Proteins; Glutathione Transferase; Polymerase Chain Reaction; Protein Binding; Protein Engineering; Recombinant Fusion Proteins; Repressor Proteins; Streptavidin
PubMed: 25560075
DOI: 10.1007/978-1-4939-2272-7_12 -
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 -
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 -
BioTechniques Apr 2019
Topics: Animals; Humans; Mass Spectrometry; Membrane Proteins; Membranes, Artificial; Optical Imaging; Protein Conformation
PubMed: 30987442
DOI: 10.2144/btn-2019-0030 -
Biochemistry and Cell Biology =... Dec 2016Membrane proteins are still heavily under-represented in the protein data bank (PDB), owing to multiple bottlenecks. The typical low abundance of membrane proteins in... (Review)
Review
Membrane proteins are still heavily under-represented in the protein data bank (PDB), owing to multiple bottlenecks. The typical low abundance of membrane proteins in their natural hosts makes it necessary to overexpress these proteins either in heterologous systems or through in vitro translation/cell-free expression. Heterologous expression of proteins, in turn, leads to multiple obstacles, owing to the unpredictability of compatibility of the target protein for expression in a given host. The highly hydrophobic and (or) amphipathic nature of membrane proteins also leads to challenges in producing a homogeneous, stable, and pure sample for structural studies. Circumventing these hurdles has become possible through the introduction of novel protein production protocols; efficient protein isolation and sample preparation methods; and, improvement in hardware and software for structural characterization. Combined, these advances have made the past 10-15 years very exciting and eventful for the field of membrane protein structural biology, with an exponential growth in the number of solved membrane protein structures. In this review, we focus on both the advances and diversity of protein production and purification methods that have allowed this growth in structural knowledge of membrane proteins through X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM).
Topics: Animals; Crystallography, X-Ray; Humans; Membrane Proteins
PubMed: 27010607
DOI: 10.1139/bcb-2015-0143 -
Bioorganic & Medicinal Chemistry Letters Feb 2020Solid-phase resins functionalized with poly-deoxythymidine (dT) oligos facilitate purification of poly-adenylated molecules from solution through high affinity, high...
Solid-phase resins functionalized with poly-deoxythymidine (dT) oligos facilitate purification of poly-adenylated molecules from solution through high affinity, high selectivity base-pairing interactions. These resins are commonly used to purify messenger RNA (mRNA) from complex biological mixtures as well as mRNA-protein fusion molecules for mRNA Display selections. Historically, dT-conjugated cellulose was the primary resin for poly-dA purification, but its scarcity has prompted the development of alternative resins, most notably dT-functionalized magnetic beads. In order to develop a cost-effective alternative to commercially available poly-dT resins for large-scale purifications of mRNA-protein fusions, we investigated the purification properties of dT-conjugated Oligo Affinity Support resin (dT-OAS) alongside poly-dT magnetic beads and dT-cellulose. dT-OAS was found to have the highest dA oligo binding capacity at 4 pmol/µg, followed by dT-magnetic beads (1.1 pmol/µg) and dT-cellulose (0.7 pmol/µg). To determine the resin specificity in the context of a complex biological mixture, we translated mRNA-protein fusions consisting of a radiolabeled Her2 affibody fused to its encoding mRNA. Commercial dT-cellulose showed the highest mRNA-affibody purification specificity, followed by dT-OAS and dT-magnetic beads. Overall, dT-OAS showed exceptionally high binding capacity and low background binding, making it an attractive alternative for large-scale mRNA purification and mRNA Display library enrichment.
Topics: Cellulose; Chromatography, Affinity; Isotope Labeling; Magnetics; Poly A; RNA, Messenger; Recombinant Fusion Proteins
PubMed: 31919017
DOI: 10.1016/j.bmcl.2019.126934 -
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