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Lab on a Chip Dec 2017Micro free-flow electrophoresis (μFFE) is a continuous separation technique in which analytes are streamed through a perpendicularly applied electric field in a planar... (Review)
Review
Micro free-flow electrophoresis (μFFE) is a continuous separation technique in which analytes are streamed through a perpendicularly applied electric field in a planar separation channel. Analyte streams are deflected laterally based on their electrophoretic mobilities as they flow through the separation channel. A number of μFFE separation modes have been demonstrated, including free zone (FZ), micellar electrokinetic chromatography (MEKC), isoelectric focusing (IEF) and isotachophoresis (ITP). Approximately 60 articles have been published since the first μFFE device was fabricated in 1994. We anticipate that recent advances in device design, detection, and fabrication, will allow μFFE to be applied to a much wider range of applications. Applications particularly well suited for μFFE analysis include continuous, real time monitoring and microscale purifications.
Topics: Cell Line; Electrophoresis; Equipment Design; Humans; Lab-On-A-Chip Devices; Microfluidic Analytical Techniques; Proteins
PubMed: 29077103
DOI: 10.1039/c7lc01105a -
Methods in Molecular Biology (Clifton,... 2020Intrinsically disordered proteins (IDPs) describe a group of proteins that do not have a regular tertiary structure and typically have very little ordered secondary...
Intrinsically disordered proteins (IDPs) describe a group of proteins that do not have a regular tertiary structure and typically have very little ordered secondary structure. Despite not following the biochemical dogma of "structure determines function" and "function determines structure," IDPs have been identified as having numerous biological functions. We describe here the steps to express and purify the intrinsically disordered stress response protein, Late embryogenesis abundant protein 3-2 from Arabidopsis thaliana (AtLEA 3-2), with N and C isotopes in E. coli, although the protocol can be adapted for any IDP with or without isotopic labeling. The atlea 3-2 gene has been cloned into the pET-SUMO vector that in addition to the SUMO portion encodes an N-terminal hexahistidine sequence (His-tag). This vector allows for the SUMO-AtLEA 3-2 fusion protein to be purified using Ni-affinity chromatography and, through the use of ubiquitin-like-specific protease 1 (Ulp1, a SUMO protease), results in an AtLEA 3-2 with a native N-terminus. We also describe the expression and purification of Ulp1 itself.
Topics: Cell Fractionation; Electrophoresis, Polyacrylamide Gel; Intrinsically Disordered Proteins; Recombinant Proteins
PubMed: 32696357
DOI: 10.1007/978-1-0716-0524-0_8 -
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 -
Methods in Molecular Biology (Clifton,... 2017The discovery of protein-protein interaction networks can lead to the unveiling of protein complex(es) forming cellular machinerie(s) or reveal component proteins of a...
The discovery of protein-protein interaction networks can lead to the unveiling of protein complex(es) forming cellular machinerie(s) or reveal component proteins of a specific cellular pathway. Deciphering protein-protein interaction networks therefore contributes to a deeper understanding of how cells function. Here we describe the protocol to perform tandem affinity purification (TAP) in bacteria, which enables the identification of the partners of a bait protein under native conditions. This method consists in two sequential steps of affinity purification using two different tags. For that purpose, the bait protein is translationally fused to the TAP tag, which consists of a calmodulin binding peptide (CBP) and two immunoglobulin G (IgG) binding domains of Staphylococcus aureus protein A (ProtA) that are separated by the tobacco etch virus (TEV) protease cleavage site. After the first round of purification based on the binding of ProtA to IgG coated beads, TEV protease cleavage releases CBP-tagged bait-protein along with its partners for a second round of purification on calmodulin affinity resin and leaves behind protein contaminants bound to IgG. Creating the TAP-tag translational fusion at the chromosomal locus allows detection of protein interactions occurring in physiological conditions.
Topics: Amino Acid Sequence; Base Sequence; Blotting, Western; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Gene Expression; Gene Order; Genetic Vectors; Immunoprecipitation; Multiprotein Complexes; Protein Binding; Protein Interaction Mapping; Proteins; Proteomics; Recombinant Fusion Proteins; Tandem Mass Spectrometry
PubMed: 28667616
DOI: 10.1007/978-1-4939-7033-9_18 -
Bioanalysis Jul 2018
Topics: Biomarkers; Chromatography, Liquid; Immunoassay; Proteins; Solid Phase Extraction; Tandem Mass Spectrometry
PubMed: 29972308
DOI: 10.4155/bio-2018-0056 -
Methods in Molecular Biology (Clifton,... 2018The availability of different polyubiquitin chains of specific linkage types has changed the appreciation of the specificity in the ubiquitin (Ub) system. Numerous E2...
The availability of different polyubiquitin chains of specific linkage types has changed the appreciation of the specificity in the ubiquitin (Ub) system. Numerous E2 Ub-conjugating enzymes and E3 Ub ligases, Ub-binding domains (UBDs), and deubiquitinases (DUBs) are now known to assemble, bind, or hydrolyze individual linkage types, respectively. Biochemical and structural studies of these processes require milligram quantities of pure polyUb. Here we describe protocols that allow the enzymatic synthesis and purification of six of the eight homotypic polyUb chains through the use of chain-specific Ub ligases and DUBs.
Topics: Humans; Mutation; Polyubiquitin; Protein Binding; Protein Interaction Domains and Motifs; Recombinant Proteins; Ubiquitin; Ubiquitin-Conjugating Enzymes; Ubiquitination
PubMed: 30242704
DOI: 10.1007/978-1-4939-8706-1_6 -
Acta Crystallographica. Section D,... Feb 2017With continuous technical improvements at synchrotron facilities, data-collection rates have increased dramatically. This makes it possible to collect diffraction data... (Review)
Review
With continuous technical improvements at synchrotron facilities, data-collection rates have increased dramatically. This makes it possible to collect diffraction data for hundreds of protein-ligand complexes within a day, provided that a suitable crystal system is at hand. However, developing a suitable crystal system can prove challenging, exceeding the timescale of data collection by several orders of magnitude. Firstly, a useful crystallization construct of the protein of interest needs to be chosen and its expression and purification optimized, before screening for suitable crystallization and soaking conditions can start. This article reviews recent publications analysing large data sets of crystallization trials, with the aim of identifying factors that do or do not make a good crystallization construct, and gives guidance in the design of an expression construct. It provides an overview of common protein-expression systems, addresses how ligand binding can be both help and hindrance for protein purification, and describes ligand co-crystallization and soaking, with an emphasis on troubleshooting.
Topics: Animals; Crystallization; Crystallography, X-Ray; Humans; Ligands; Models, Molecular; Mutation; Protein Conformation; Protein Engineering; Proteins
PubMed: 28177304
DOI: 10.1107/S2059798316020271 -
Molecules (Basel, Switzerland) Jan 2022The chaperone DNAJB6b delays amyloid formation by suppressing the nucleation of amyloid fibrils and increases the solubility of amyloid-prone proteins. These dual...
The chaperone DNAJB6b delays amyloid formation by suppressing the nucleation of amyloid fibrils and increases the solubility of amyloid-prone proteins. These dual effects on kinetics and equilibrium are related to the unusually high chemical potential of DNAJB6b in solution. As a consequence, the chaperone alone forms highly polydisperse oligomers, whereas in a mixture with an amyloid-forming protein or peptide it may form co-aggregates to gain a reduced chemical potential, thus enabling the amyloid peptide to increase its chemical potential leading to enhanced solubility of the peptide. Understanding such action at the level of molecular driving forces and detailed structures requires access to highly pure and sequence homogeneous DNAJB6b with no sequence extension. We therefore outline here an expression and purification protocol of the protein "as is" with no tags leading to very high levels of pure protein based on its physicochemical properties, including size and charge. The versatility of the protocol is demonstrated through the expression of an isotope labelled protein and seven variants, and the purification of three of these. The activity of the protein is bench-marked using aggregation assays. Two of the variants are used to produce a palette of fluorescent DNAJB6b labelled at an engineered N- or C-terminal cysteine.
Topics: Ammonium Sulfate; Amyloidogenic Proteins; Chemical Precipitation; Chromatography, Gel; Escherichia coli; Fluorescent Dyes; HSP40 Heat-Shock Proteins; Humans; Hydrogen-Ion Concentration; Molecular Chaperones; Nerve Tissue Proteins; Protein Denaturation; Protein Engineering; Recombinant Proteins; Rhodamines; Solubility; Sulfonic Acids
PubMed: 35056736
DOI: 10.3390/molecules27020418 -
Methods in Molecular Biology (Clifton,... 2017Affinity chromatography permits the isolation of a target analyte from a complex mixture and can be utilized to purify proteins, carbohydrates, drugs, haptens, or any...
Affinity chromatography permits the isolation of a target analyte from a complex mixture and can be utilized to purify proteins, carbohydrates, drugs, haptens, or any analyte of interest once an affinity pair is available. It involves the exploitation of specific interactions between a binding affinity pair, such as those between an antibody and its associated antigen, or between any ligand and its associated binding receptor/protein. With the discovery of protein A in 1970, and, subsequently protein G and L, immuno-affinity chromatography has grown in popularity and is now the standard methodology for the purification of antibodies which may be implemented for a selection of different applications such as immunodiagnostics. This chapter is designed to inform the researcher about the basic techniques involved in the affinity chromatography-based purification of monoclonal, polyclonal, and recombinant antibodies. Examples are provided for the use of protein A and G. In addition, tables are provided that allow the reader to select the most appropriate protein for use in the isolation of their antibody.
Topics: Animals; Antibodies; Antibodies, Monoclonal; Bacterial Proteins; Chemical Precipitation; Chromatography, Affinity; Humans; Staphylococcal Protein A
PubMed: 27730559
DOI: 10.1007/978-1-4939-6412-3_15 -
Methods in Enzymology 2021Nicotinic acetylcholine receptors are members of the Cys-loop superfamily of pentameric ligand-gated ion channels. The electric organ of the Torpedo ray is...
Nicotinic acetylcholine receptors are members of the Cys-loop superfamily of pentameric ligand-gated ion channels. The electric organ of the Torpedo ray is extraordinarily rich in an acetylcholine receptor that is homologous to the human nicotinic receptor found at the neuromuscular junction. Due to this abundant natural source in the fish and the relatively accessible preparation of the neuromuscular junction (compared to a central synapse), this muscle-type receptor and specifically the fish receptors have long been used as the prototype for study of nicotinic receptors. However, an absence of structural detail at high resolution has limited the chemical interpretation of this archetypal nicotinic receptor. One of the main concerns in preparing receptor for high resolution structural analysis was its documented sensitivity to particular detergents and requirements for specific lipids in order to maintain function after reconstitution in a membrane. Here, we present methods for purifying native nicotinic receptor from Torpedo electric tissue that maintains functionality after reconstitution and that is amenable to high resolution structural analysis. The specific developments we describe include detergent exchange during purification, inclusion of specific lipids during purification and for nanodisc reconstitution, and synthesis of a new affinity reagent for rapid isolation of receptors.
Topics: Animals; Fish Proteins; Ligand-Gated Ion Channels; Receptors, Nicotinic; Torpedo
PubMed: 34099171
DOI: 10.1016/bs.mie.2020.12.003