Showing papers on "Affinity chromatography published in 2015"

Purification of GST-Tagged Proteins

Abstract: Ni-NTA affinity purification of His-tagged proteins is a bind-wash-elute procedure that can be performed under native or denaturing conditions. Here, protocols for purification of His-tagged proteins under native, as well as under denaturing conditions, are given. The choice whether to purify the target protein under native or denaturing conditions depends on protein location and solubility, the accessibility of the His tag, and the desired downstream application. His-tagged proteins can be purified by a single-step affinity chromatography, namely immobilized metal ion affinity chromatography (IMAC), which is commercially available in different kinds of formats, Ni-NTA matrices being the most widely used. The provided protocols describe protein purification in the batch binding mode and apply gravity-assisted flow in disposable columns; this procedure is simple to conduct and extremely robust. IMAC purification can equally be performed in prepacked columns using FPLC or other liquid chromatography instrumentation, or using magnetic bead-based methods (Block et al., 2009).

Antibody Fragments and Their Purification by Protein L Affinity Chromatography

Abstract: Antibodies and related proteins comprise one of the largest and fastest-growing classes of protein pharmaceuticals. A majority of such molecules are monoclonal antibodies; however, many new entitie ...

Selective Proteomic Proximity Labeling Assay Using Tyramide (SPPLAT): A Quantitative Method for the Proteomic Analysis of Localized Membrane-Bound Protein Clusters

Abstract: This manuscript describes a new and general method to identify proteins localized into spatially restricted membrane microenvironments. Horseradish peroxidase (HRP) is brought into contact with a target protein by being covalently linked to a primary or secondary antibody, an antigen or substrate, a drug, or a toxin. A biotinylated tyramide-based reagent is then added. In the presence of HRP and hydrogen peroxide, the reagent is converted into a free radical that only diffuses a short distance before covalently labeling proteins within a few tens to hundreds of nanometers from the target. The biotinylated proteins can then be isolated by standard affinity chromatography and identified by liquid chromatography (LC) and mass spectrometry (MS). The assay can be made quantitative by using stable isotope labeling with amino acids in cell culture (SILAC) or isobaric tagging at the peptide level. © 2017 by John Wiley & Sons, Inc.

Purification, refolding, and characterization of recombinant human paraoxonase-1

Abstract: A high density lipoprotein (HDL)-linked enzyme with antioxidant and antiatherogenic properties, paraoxonase-1(PON1), prevents the formation of atherosclerotic lesions in humans. In the present study, a recombinant hPON1 gene was produced using a small ubiquitin-related modifier (SUMO) fusion protein expression system. To that end, the hPON1 gene was amplified from human liver-ready cDNA, cloned into the expression vector pET SUMO, and expressed in Escherichia coli BL21 (DE3). The predominance of the expressed fusion SUMO-hPON1 protein was inclusion bodies and purified using 6xHis affinity chromatography under natural and denaturing conditions. Subsequently, the enzyme was purified and refolded directly on the affinity matrix under redox conditions to obtain a bioactive protein in a single step. The inclusion bodies were solubilized with urea, guanidine hydrochloride, and Triton X-100 and refolded in vitro. After purification, 0.045 mg/mL protein in soluble fraction and 0.108 mg/mL protein from inclusion bodies were obtained. Optimum temperature, pH, and ionic strength for rhPON1 activity were determined as 40 $^{\circ}$C, 10.0, and 100 mM, respectively. The kinetic parameters K$_{m}$ and V$_{\max}$ for rhPON1 were determined as 0.94 mM and 110.01 EU/mL, respectively, by using Lineweaver-Burk plots.

Modular microfluidics for point-of-care protein purifications

Abstract: Biochemical separations are the heart of diagnostic assays and purification methods for biologics. On-chip miniaturization and modularization of separation procedures will enable the development of customized, portable devices for personalized health-care diagnostics and point-of-use production of treatments. In this report, we describe the design and fabrication of miniature ion exchange, size exclusion and affinity chromatography modules for on-chip clean-up of recombinantly-produced proteins. Our results demonstrate that these common separations techniques can be implemented in microfluidic modules with performance comparable to conventional approaches. We introduce embedded 3-D microfluidic interconnects for integrating micro-scale separation modules that can be arranged and reconfigured to suit a variety of fluidic operations or biochemical processes. We demonstrate the utility of the modular approach with a platform for the enrichment of enhanced green fluorescent protein (eGFP) from Escherichia coli lysate through integrated affinity and size-exclusion chromatography modules.

Quantitative affinity purification mass spectrometry: a versatile technology to study protein-protein interactions.

Abstract: While the genomic revolution has dramatically accelerated the discovery of disease-associated genes, the functional characterization of the corresponding proteins lags behind. Most proteins fulfill their tasks in complexes with other proteins, and analysis of protein-protein interactions (PPIs) can therefore provide insights into protein function. Several methods can be used to generate large-scale protein interaction networks. However, most of these approaches are not quantitative and therefore cannot reveal how perturbations affect the network. Here, we illustrate how a clever combination of quantitative mass spectrometry with different biochemical methods provides a rich toolkit to study different aspects of PPIs including topology, subunit stoichiometry, and dynamic behavior.

Mass spectrometric analysis of protein tyrosine nitration in aging and neurodegenerative diseases.

Abstract: This review highlights the significance of protein tyrosine nitration (PTN) in signal transduction pathways, the progress achieved in analytical methods, and the implication of nitration in the cellular pathophysiology of aging and age-related neurodegenerative diseases. Although mass spectrometry of nitrated peptides has become a powerful tool for the characterization of nitrated peptides, the low stoichiometry of this modification clearly necessitates the use of affinity chromatography to enrich modified peptides. Analysis of nitropeptides involves identification of endogenous, intact modification as well as chemical conversion of the nitro group to a chemically reactive amine group and further modifications that enable affinity capture and enhance detectability by altering molecular properties. In this review, we focus on the recent progress in chemical derivatization of nitropeptides for enrichment and mass analysis, and for detection and quantification using various analytical tools. PTN participates in physiological processes, such as aging and neurodegenerative diseases. Accumulation of 3-nitrotyrosine has been found to occur during the aging process; this was identified through mass spectrometry. Further, there are several studies implicating the presence of nitrated tyrosine in age-related diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis.

Recombinant Production of Snakin-2 (an Antimicrobial Peptide from Tomato) in E. coli and Analysis of Its Bioactivity

Abstract: Antimicrobial peptides (AMPs) represent a diverse group of biologically active molecules that are part of the innate immune systems of a variety of organisms. Their primary function consists of protecting the host organism against invading microorganisms, including pathogens. AMPs show a broad spectrum of secondary structures, which are essential for antimicrobial activity. In this study, we produced snakin-2 (SN2), a 66-amino-acid-(aa)-long AMP from Solanum lycopersicum as a recombinant protein in E. coli. This AMP belongs to the GASA/GAST protein family and possesses a highly conserved 60-aa-long domain with six disulfide bonds in the C-terminus of the peptide. Because of the toxicity of SN2 against its producing E. coli strain, the AMP was attached to an N-terminal fusion protein (thioredoxin A), which was removed after affinity chromatography purification. The total yield of recombinant SN2 was approximately 1 mg/L. The membrane-active SN2 showed a bactericidal and fungicidal bioactivity, which can be explained by perforation of biomembranes of bacteria and fungi.

AmyM, a Novel Maltohexaose-Forming α-Amylase from Corallococcus sp. strain EGB.

Abstract: A novel α-amylase, AmyM, was purified from the culture supernatant of Corallococcus sp. strain EGB. AmyM is a maltohexaose-forming exoamylase with an apparent molecular mass of 43 kDa. Based on the results of matrix-assisted laser desorption ionization-time of flight mass spectrometry and peptide mass fingerprinting of AmyM and by comparison to the genome sequence of Corallococcus coralloides DSM 2259, the AmyM gene was identified and cloned into Escherichia coli. amyM encodes a secretory amylase with a predicted signal peptide of 23 amino acid residues, which showed no significant identity with known and functionally verified amylases. amyM was expressed in E. coli BL21(DE3) cells with a hexahistidine tag. The signal peptide efficiently induced the secretion of mature AmyM in E. coli. Recombinant AmyM (rAmyM) was purified by Ni-nitrilotriacetic acid (NTA) affinity chromatography, with a specific activity of up to 14,000 U/mg. rAmyM was optimally active at 50°C in Tris-HCl buffer (50 mM; pH 7.0) and stable at temperatures of <50°C. rAmyM was stable over a wide range of pH values (from pH 5.0 to 10.0) and highly tolerant to high concentrations of salts, detergents, and various organic solvents. Its activity toward starch was independent of calcium ions. The Km and Vmax of recombinant AmyM for soluble starch were 6.61 mg ml(-1) and 44,301.5 μmol min(-1) mg(-1), respectively. End product analysis showed that maltohexaose accounted for 59.4% of the maltooligosaccharides produced. These characteristics indicate that AmyM has great potential in industrial applications.

Characterisation of a novel thermostable endoglucanase from Alicyclobacillus vulcanalis of potential application in bioethanol production

Abstract: A novel endoglucanase encoding gene was cloned from Alicyclobacillus vulcanalis and expressed in E. coli. The deduced amino acid sequence showed highest identity with α-l-arabinofuranosidase-like proteins from glycoside hydrolase family 51. The recombinant enzyme was purified by affinity chromatography and characterised in terms of its potential suitability for lignocellulose hydrolysis at high temperature in the production of bioethanol. The purified enzyme displayed maximum activity at 80 °C and pH 3.6–4.5. Tween 20 was found to have a beneficial effect on enzyme activity and thermal stability. When incubated in the presence of 0.1 % Tween 20, the enzyme retained full activity after 72 h at 70 °C and 78 % of original activity after 72 h at 75 °C. Maximum activity was observed on carboxymethyl cellulose, and the purified enzyme also hydrolysed lichenan, barley β-glucan and xylan. The purified enzyme decreased the viscosity of carboxymethyl cellulose when assessed at 70–85 °C and was capable of releasing reducing sugars from acid-pretreated straw at 70 and 75 °C. The results indicate the potential suitability of the enzyme for industrial application in the production of cellulosic bioethanol.

Identification of an adeno-associated virus binding epitope for AVB sepharose affinity resin

Abstract: Recent successes of adeno-associated virus (AAV)-based gene therapy have created a demand for large-scale AAV vector manufacturing and purification techniques for use in clinical trials and beyond. During the development of purification protocols for rh.10, hu.37, AAV8, rh.64R1, AAV3B, and AAV9 vectors, based on a widely used affinity resin, AVB sepharose (GE), we found that, under the same conditions, different serotypes have different affinities to the resin, with AAV3B binding the best and AAV9 the poorest. Further analysis revealed a surface-exposed residue (amino acid number 665 in AAV8 VP1 numbering) differs between the high-affinity AAV serotypes (serine in AAV3B, rh.10, and hu.37) and the low-affinity ones (asparagine in AAV8, rh.64R1, and AAV9). The residue locates within a surface-exposed, variable epitope flanked by highly conserved residues. The substitution of the epitope in AAV8, rh.64R1, and AAV9 with the corresponding epitope of AAV3B (SPAKFA) resulted in greatly increased affinity to AVB sepharose with no reduction in the vectors' in vitro potency. The presence of the newly identified AVB-binding epitope will be useful for affinity resin selection for the purification of novel AAV serotypes. It also suggests the possibility of vector engineering to yield a universal affinity chromatography purification method for multiple AAV serotypes.

Chapter Nine - Purification of GST-Tagged Proteins

Abstract: This protocol describes the purification of recombinant proteins fused to glutathione S-transferase (GST, GST-tagged proteins) by Glutathione Affinity purification. The GST tag frequently increases the solubility of the fused protein of interest and thus enables its purification and subsequent functional characterization. The GST-tagged protein specifically binds to glutathione immobilized to a matrix (e.g., agarose) and can be easily separated from a cell lysate by a bind-wash-elute procedure. GST-tagged proteins are often used to study protein–protein interactions, again making use of glutathione affinity in a procedure called a GST pull-down assay. The protocol is designed to process 200 ml of E. coli culture expressing intermediate to high amounts of a GST-tagged protein (~ 25 mg l− 1). Depending on the expression rate or the available culture volume, the scale can be increased or decreased linearly. The protocol can also be used to purify GST-tagged proteins from other expression systems, such as insect or mammalian cells. Tips are provided to aid in modifying certain steps if proteins shall be recovered from alternative expression systems.

Endogenous biotin‐binding proteins: an overlooked factor causing false positives in streptavidin‐based protein detection

Abstract: Biotinylation is widely used in DNA, RNA and protein probing assays as this molecule has generally no impact on the biological activity of its substrate. During the streptavidin-based detection of glycoproteins in Lactobacillus rhamnosus GG with biotinylated lectin probes, a strong positive band of approximately 125 kDa was observed, present in different cellular fractions. This potential glycoprotein reacted heavily with concanavalin A (ConA), a lectin that specifically binds glucose and mannose residues. Surprisingly, this protein of 125 kDa could not be purified using a ConA affinity column. Edman degradation of the protein, isolated via cation and anion exchange chromatography, lead to the identification of the band as pyruvate carboxylase, an enzyme of 125 kDa that binds biotin as a cofactor. Detection using only the streptavidin conjugate resulted in more false positive signals of proteins, also in extracellular fractions, indicating biotin-associated proteins. Indeed, biotin is a known cofactor of numerous carboxylases. The potential occurence of false positive bands with biotinylated protein probes should thus be considered when using streptavidin-based detection, e.g. by developing a blot using only the streptavidin conjugate. To circumvent these false positives, alternative approaches like detection based on digoxigenin labelling can also be used.

A Chlorogenic Acid Esterase with a Unique Substrate Specificity from Ustilago maydis

Abstract: An extracellular chlorogenic acid esterase from Ustilago maydis (UmChlE) was purified to homogeneity by using three separation steps, including anion-exchange chromatography on a Q Sepharose FF column, preparative isoelectric focusing (IEF), and, finally, a combination of affinity chromatography and hydrophobic interaction chromatography on polyamide. SDS-PAGE analysis suggested a monomeric protein of ∼71 kDa. The purified enzyme showed maximal activity at pH 7.5 and at 37°C and was active over a wide pH range (3.5 to 9.5). Previously described chlorogenic acid esterases exhibited a comparable affinity for chlorogenic acid, but the enzyme from Ustilago was also active on typical feruloyl esterase substrates. Kinetic constants for chlorogenic acid, methyl p-coumarate, methyl caffeate, and methyl ferulate were as follows: Km values of 19.6 μM, 64.1 μM, 72.5 μM, and 101.8 μM, respectively, and kcat/Km values of 25.83 mM(-1) s(-1), 7.63 mM(-1) s(-1), 3.83 mM(-1) s(-1) and 3.75 mM(-1) s(-1), respectively. UmChlE released ferulic, p-coumaric, and caffeic acids from natural substrates such as destarched wheat bran (DSWB) and coffee pulp (CP), confirming activity on complex plant biomass. The full-length gene encoding UmChlE consisted of 1,758 bp, corresponding to a protein of 585 amino acids, and was functionally produced in Pichia pastoris GS115. Sequence alignments with annotated chlorogenic acid and feruloyl esterases underlined the uniqueness of this enzyme.

High yield expression of novel glutaminase free l -asparaginase II of Pectobacterium carotovorum MTCC 1428 in Bacillus subtilis WB800N

Abstract: Gene encoding glutaminase-free L-asparaginase II (ans B2) from Pectobacterium carotovorum MTCC 1428 was cloned into pHT43, transformed in Bacillus subtilis WB800N and optimised the expression levels of recombinant enzyme. A three-fold higher enzyme production was observed with an efficient transformant as compared to native strain. Enzyme localization studies revealed that >90% of recombinant enzyme is secreted extracellularly, a little fraction is attached to the membrane (>6%) and localised intracellularly (3%). The expression of recombinant L-asparaginase II was confirmed by SDS-PAGE, IMAC (Immobilised metal ion affinity chromatography) purification followed by Western blotting. Process parameter optimization with OFAT (one factor at a time) revealed that rpm (120), temperature (37 °C), Isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration (1 mM) and time of induction (0.8 OD600nm) plays a vital role where a maximum of 55 IU/ml was achieved. Further, consecutive induction by IPTG improved the enzyme production up to 105 IU/ml with a specific activity of 101 IU/mg of protein. Molecular modelling analysis depicted that amino acids, GLY60, GLY119 and ALA252 in the active site are responsible for the glutaminase free L-asparaginase II activity. This is the first report on enhanced expression of recombinant glutaminase-free L-asparaginase II by intermediate addition of IPTG.

Affinity Chromatography: A Historical Perspective

Abstract: Affinity chromatography is one of the most selective and versatile forms of liquid chromatography for the separation or analysis of chemicals in complex mixtures. This method makes use of a biologically related agent as the stationary phase, which provides an affinity column with the ability to bind selectively and reversibly to a given target in a sample. This review examines the early work in this method and various developments that have lead to the current status of this technique. The general principles of affinity chromatography are briefly described as part of this discussion. Past and recent efforts in the generation of new binding agents, supports, and immobilization methods for this method are considered. Various applications of affinity chromatography are also summarized, as well as the influence this field has played in the creation of other affinity-based separation or analysis methods.