-
Journal of Chromatography. B,... Mar 2007Staphylococcal protein A (SPA) is one of the first discovered immunoglobulin binding molecules and has been extensively studied during the past decades. Due to its... (Review)
Review
Staphylococcal protein A (SPA) is one of the first discovered immunoglobulin binding molecules and has been extensively studied during the past decades. Due to its affinity to immunoglobulins, SPA has found widespread use as a tool in the detection and purification of antibodies and the molecule has been further developed to one of the most employed affinity purification systems. Interestingly, a minimized SPA derivative has been constructed and a domain originating from SPA has been improved to withstand the harsh environment employed in industrial purifications. This review will focus on the development of different affinity molecules and matrices for usage in antibody purification.
Topics: Antibodies; Chromatography, Affinity; Protein Engineering; Staphylococcal Protein A
PubMed: 17030158
DOI: 10.1016/j.jchromb.2006.09.030 -
Methods (San Diego, Calif.) Jul 2001Identification of components present in biological complexes requires their purification to near homogeneity. Methods of purification vary from protein to protein,... (Review)
Review
Identification of components present in biological complexes requires their purification to near homogeneity. Methods of purification vary from protein to protein, making it impossible to design a general purification strategy valid for all cases. We have developed the tandem affinity purification (TAP) method as a tool that allows rapid purification under native conditions of complexes, even when expressed at their natural level. Prior knowledge of complex composition or function is not required. The TAP method requires fusion of the TAP tag, either N- or C-terminally, to the target protein of interest. Starting from a relatively small number of cells, active macromolecular complexes can be isolated and used for multiple applications. Variations of the method to specifically purify complexes containing two given components or to subtract undesired complexes can easily be implemented. The TAP method was initially developed in yeast but can be successfully adapted to various organisms. Its simplicity, high yield, and wide applicability make the TAP method a very useful procedure for protein purification and proteome exploration.
Topics: Bacterial Proteins; Blotting, Western; DNA, Bacterial; Fungal Proteins; Genetic Vectors; Methods; Mutation; Polymerase Chain Reaction; Proteins; Proteome; Ribonucleases; Ribonucleoproteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Staphylococcus aureus
PubMed: 11403571
DOI: 10.1006/meth.2001.1183 -
Protein Expression and Purification Dec 2015Availability of highly purified proteins in quantity is crucial for detailed biochemical and structural investigations. Fusion tags are versatile tools to facilitate...
Availability of highly purified proteins in quantity is crucial for detailed biochemical and structural investigations. Fusion tags are versatile tools to facilitate efficient protein purification and to improve soluble overexpression of proteins. Various purification and fusion tags have been widely used for overexpression in Escherichia coli. However, these tags might interfere with biological functions and/or structural investigations of the protein of interest. Therefore, an additional purification step to remove fusion tags by proteolytic digestion might be required. Here, we describe a set of new vectors in which yeast SUMO (SMT3) was used as the highly specific recognition sequence of ubiquitin-like protease 1, together with other commonly used solubility enhancing proteins, such as glutathione S-transferase, maltose binding protein, thioredoxin and trigger factor for optimizing soluble expression of protein of interest. This tandem SUMO (T-SUMO) fusion system was tested for soluble expression of the C-terminal domain of TonB from different organisms and for the antiviral protein scytovirin.
Topics: Bacterial Proteins; Base Sequence; Carrier Proteins; Cloning, Molecular; Cyanobacteria; Cysteine Endopeptidases; Escherichia coli; Genetic Vectors; Helicobacter pylori; Lectins; Membrane Proteins; Proteolysis; Pseudomonas; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Small Ubiquitin-Related Modifier Proteins; Solubility
PubMed: 26297996
DOI: 10.1016/j.pep.2015.08.019 -
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 Enzymology 2014This protocol describes a denaturing polyacrylamide gel system utilizing sodium dodecyl sulfate (SDS) to separate protein molecules based on size as first described by...
This protocol describes a denaturing polyacrylamide gel system utilizing sodium dodecyl sulfate (SDS) to separate protein molecules based on size as first described by Laemmli (1970). SDS-PAGE can be used to monitor protein purifications, check the purity of samples, and to estimate molecular weights for unknown proteins.
Topics: Electrophoresis, Polyacrylamide Gel; Proteins
PubMed: 24674069
DOI: 10.1016/B978-0-12-420119-4.00012-4 -
Journal of Chromatography Oct 1979This review attempts to identify proteins which selectively interact with immobilised triazine dyes such as Cibacron blue F3GA and Procion red HE 3B. Different support... (Review)
Review
This review attempts to identify proteins which selectively interact with immobilised triazine dyes such as Cibacron blue F3GA and Procion red HE 3B. Different support matrices are compared by examining the capacities of these dyes for proteins. Various approaches to the immobilisation of triazine dyes are considered together with the use of spacers. Some theories of the mechanism of protein retardation by immobilised dyes are discussed. A number of methods are suggested for the measurement of dye concentrations and for the modification of the binding of proteins to dye columns. The variety of elution methods is compared with a view to optimizing purifications. The scope of applications is reviewed as well as the choice of dye. Some advantages of triazine dyes over other affinity ligands are given. It is concluded that although no satisfactory mechanism for the binding of triazine dyes to proteins has yet been proposed, these dyes possess considerable potential for protein purification, particularly when applied on the large scale.
Topics: Animals; Chromatography, Affinity; Coloring Agents; Enzymes; Humans; Indicators and Reagents; Proteins; Triazines
PubMed: 395164
DOI: 10.1016/s0021-9673(00)88187-x -
Journal of Chromatography. A Sep 2018Recently, the importance of biopharmaceuticals in medical treatments has been increasing, and effective protein purification methods are strongly required for their...
Recently, the importance of biopharmaceuticals in medical treatments has been increasing, and effective protein purification methods are strongly required for their production. In the present study, a temperature-responsive solid-phase extraction (SPE) column was developed for the purification of proteins without affecting their bioactivity. A temperature-responsive polymer hydrogel-modified stationary phase was prepared by coating aminopropyl silica beads (average diameter, 40-64 μm) with poly(N-isopropylacrylamide) (PNIPAAm)-based thermoresponsive hydrogels. n-Butyl methacrylate and acrylic acid were copolymerized with PNIPAAm as hydrophobic and anionic monomers, respectively. Using these temperature-responsive SPE columns, targeted proteins were retained on the thermoresponsive hydrogel at 40 °C through hydrophobic and electrostatic interactions. After the temperature was reduced from 40 °C to 4 °C, the retained proteins were successfully eluted from the column. Using the temperature-responsive SPE system, lysozyme was successfully separated from ovalbumin without any loss in bioactivity (99.7 ± 0.1%). Rituximab, a monoclonal antibody, was also purified from BSA or hybridoma cell culture medium using the prepared SPE column. Denaturation of rituximab was not observed in the rituximab fraction eluted from the SPE column. These results demonstrate that temperature-responsive polymer-based SPE can be applied in biomedical purifications, while maintaining the biological activity of the proteins.
Topics: Acrylic Resins; Chemistry Techniques, Analytical; Chromatography, Affinity; Hydrogels; Hydrophobic and Hydrophilic Interactions; Muramidase; Polymerization; Polymers; Proteins; Silicon Dioxide; Solid Phase Extraction; Static Electricity; Temperature
PubMed: 30033167
DOI: 10.1016/j.chroma.2018.07.027 -
Advances in Chromatography 1989Recently Giddings discussed the prospect of combining two separation mechanisms in such a way that when "a sample is subjected to two displacement processes oriented at... (Review)
Review
Recently Giddings discussed the prospect of combining two separation mechanisms in such a way that when "a sample is subjected to two displacement processes oriented at right angles to one another" a two-dimensional separation is carried out. In this review I have focused attention on the various ramifications of this concept in terms of combining two or more chromatographic techniques on-line to conduct MDLC for the purpose of purifying proteins. In general, the MDLC approaches discussed here were classified into two major categories. The first category involves the placement of several separation mechanisms in the same chromatographic work space (the chromatographic column). In this case the displacement processes are collinear. It is hoped that these new chromatographic packings and columns will display surface characteristics capable of a wide range of highly discriminating selectivities that can be modulated by mobile-phase changes to a greater extent than the nonspecific chromatographic techniques such as IEC and HIC. The ability to modulate the mobile phase to generate new selectivities is important in expanding the usefulness of these packings in comparison to the very high selectivity of affinity chromatography, which usually has little use outside its initial intended purpose to purify a particular protein. The second category involves the on-line physical coupling of two or more chromatography columns, each packed with a different chromatography material. Again the idea is to create or design a simple self-contained system that is capable of generating a wide range of high selectivities. Indeed, the on-line coupling of two different chromatographic packings for the purification of a single protein represents a line trace through "a discrete independent 2-dimensional system". These systems are highly attractive in large-scale purification, especially when using "on-off" chromatography, which eliminates the need for sophisticated gradient elution hardware. The purification of a single protein from its biological matrix is usually a multidimensional process utilizing several different separation technologies. By nature this leads to lengthy purifications that are frequently labor-intensive and expensive to scale up. It is my belief that future developments in the concept of on-line MDLC techniques involving complex chromatographic materials and column coupling will merge to create significant improvements in the protein purification process.
Topics: Chemical Phenomena; Chemistry; Chromatography, Liquid; Proteins
PubMed: 2667288
DOI: No ID Found -
Molecular & Cellular Proteomics : MCP Jun 2011Recent large-scale data sets of protein complex purifications have provided unprecedented insights into the organization of cellular protein complexes. Several... (Comparative Study)
Comparative Study
Recent large-scale data sets of protein complex purifications have provided unprecedented insights into the organization of cellular protein complexes. Several computational methods have been developed to detect co-complexed proteins in these data sets. Their common aim is the identification of biologically relevant protein complexes. However, much less is known about the network of direct physical protein contacts within the detected protein complexes. Therefore, our work investigates whether direct physical contacts can be computationally derived by combining raw data of large-scale protein complex purifications. We assess four established scoring schemes and introduce a new scoring approach that is specifically devised to infer direct physical protein contacts from protein complex purifications. The physical contacts identified by the five methods are comprehensively benchmarked against different reference sets that provide evidence for true physical contacts. Our results show that raw purification data can indeed be exploited to determine high-confidence physical protein contacts within protein complexes. In particular, our new method outperforms competing approaches at discovering physical contacts involving proteins that have been screened multiple times in purification experiments. It also excels in the analysis of recent protein purification screens of molecular chaperones and protein kinases. In contrast to previous findings, we observe that physical contacts inferred from purification experiments of protein complexes can be qualitatively comparable to binary protein interactions measured by experimental high-throughput assays such as yeast two-hybrid. This suggests that computationally derived physical contacts might complement binary protein interaction assays and guide large-scale interactome mapping projects by prioritizing putative physical contacts for further experimental screens.
Topics: Algorithms; Computer Simulation; Data Interpretation, Statistical; Models, Molecular; Multiprotein Complexes; Phosphoric Monoester Hydrolases; Phosphotransferases; Protein Interaction Domains and Motifs; Protein Interaction Mapping; Saccharomyces cerevisiae Proteins
PubMed: 21451165
DOI: 10.1074/mcp.M110.004929 -
Methods in Enzymology 2009Prior to embarking upon the purification of a protein, one should begin by considering what the protein is to be used for. In particular, how much of the protein is... (Review)
Review
Prior to embarking upon the purification of a protein, one should begin by considering what the protein is to be used for. In particular, how much of the protein is needed, what should be its state of purity, and must it be folded correctly and associated with various other peptides or cofactors. Using such criteria, an appropriate assay should be chosen and a procedure be planned taking into account the source of the protein, how it is to be extracted from the source, and what agents the protein ought to be exposed to or ultimately be stored in. One is often surprised at the time necessary to develop an appropriate protein purification procedure relative to the time required to clone a gene or to accumulate information with the purified protein. There are an overwhelming number of options for protein purification steps, so forethought is necessary to expedite the tedious job of developing the purification scheme, or to avoid having to redesign it upon attempting to use the protein. This chapter points out general considerations to be undertaken in designing, organizing, and executing the purification, while subsequent chapters of this volume supply more specific options and technical details.
Topics: Animals; Clinical Laboratory Techniques; Humans; Preservation, Biological; Proteins; Recombinant Proteins; Transfection
PubMed: 19892162
DOI: 10.1016/S0076-6879(09)63002-0