Use Of Affinity Chromatography Research Articles

Simple Low-Cost Production of DNA MS2 Virus-Like Particles As Molecular Diagnostic Controls.

Suitable controls are integral for the validation and continued quality assurance of diagnostic workflows. Plasmids, DNA, or in vitro transcribed RNA are often used to validate novel diagnostic workflows, however, they are poorly representative of clinical samples. RNA phage virus-like particles (VLPs) packaged with exogenous RNA have been used in clinical diagnostics as workflow controls, serving as surrogates for infectious viral particles. Comparable controls for DNA viruses are more challenging to produce, with analogous DNA phages being infectious and packaging of DNA within RNA phages requiring complex purification procedures and expensive chemical linkers. We present a simple and inexpensive method to produce Emesvirus zinderi (MS2) VLPs, packaged with DNA, that makes use of affinity chromatography for purification and enzymatic production of exogenous DNA suitable for packaging. The produced VLPs were packaged with hepatitis B virus DNA and were then quantified using droplet digital PCR and calibrated against the WHO international standard using a commercial assay in an accredited clinical laboratory.

Affinity Chromatography: An Enabling Technology for Large Scale Bioprocessing - How does it get to the Developing World

Affinity chromatography was initially used to describe chromatographic biological interactions such as lectin-glycoprotein, antibody -antigen and enzyme-inhibitor. This definition has expanded to include the specific interaction between a target and a ligand. The use of affinity chromatography has reached a zenith with the explosion of Mab therapeutics and the use of Protein-A chromatography for antibody capture. Now, affinity chromatography has moved to non Mab proteins. This can result in the same economic advantages as Mab, by enabling the standardization of process development and manufacturing processes in flexible multiproduct production sites. The output is improved product throughput, higher target recoveries, and potentially less expensive drugs. These advantages are available to the developed world but how do we make this technology available to the developing world?

Highly Specific Mouse Anti-Joining Chain of Human Immunoglobulin A.

Immunoglobulin A (IgA) antibodies are critical to mucosal protection, specifically dimeric IgA (dIgA) and secretory IgA (sIgA), which rely on the J chain to polymerize. There is an absence of monoclonal antibodies that can specifically bind to polymeric IgA without the need to denature the molecule. We generated a panel of highly specific mouse anti-J chain antibodies that react with both intact and denatured nonhuman primate dIgA and human dIgA and sIgA of both the IgA1 and IgA2 subclass. We expanded use of this antibody for quantification of dIgA and sIgA using biolayer interferometry or enzyme-linked immunosorbent assay and use for affinity chromatography. This is a significant improvement over available anti-IgA antibodies in the field, which will allow for expanded use in clinical testing.

Preparation and characterization of large-format macroporous cryogel disks for use in affinity chromatography and biotechnological applications.

We have prepared and evaluated larger format phage-bound epoxy-cryogel columns in order to increase the yield of bound target. Freezing thermograms showed that larger column formats (2.5-5cm diameter) are not usable due to irregular polymerization phenomena. Preparing thin disks of 0.5cm height with similar diameter proved to be an excellent alternative. Disks could be stacked and run in a chromatographic setup. In this way, we could increase the matrix volume, ligand-binding capacity, and finally the yield of bound target. By increasing the column volume about sevenfold, we observed a 12-fold increase of ligand density and a sevenfold increase in the yield of protein recovery in a column where phages were attached without spacer and a 10- to 34-fold increase in a spacer column, depending on the spacer used.

Affinity chromatography for vaccines manufacturing: Finally ready for prime time?

Affinity chromatography is among the most powerful separation techniques, achieving the finest separation with high yields even in the most challenging feed streams. Incorporating affinity chromatography in vaccine purification has long been attempted by researchers to improve unit yield and purity with the secondary goal of reducing the number of downstream process operations. Despite the success in laboratory-scale proof of concept, implementation of this technique in pilot or cGMP manufacturing has rarely been realised due to technical and economic challenges in design and manufacturing of ideal ligands as well as availability of high-productivity chromatography media. This paper reviews evolving technologies in engineered ligands and chromatography media that are encouraging companies to re-visit the possible use of affinity chromatography in larger scale vaccine purification. It is postulated that commercial-scale implementation of high throughput single-use affinity chromatography can significantly simplify process architecture, improve productivity and flexibility, and reduce cost of goods.

Studies on the purification of antibody fragments

Abstract Antigen-binding fragments (Fabs) are becoming increasingly prevalent as an alternative to full-length monoclonal antibodies. In this work, the digestion of a mixture of different human antibodies isotypes was optimized in terms of different parameters, including the concentration of papain and cysteine, and the digestion time. The recovery of the Fab fragments was subsequently evaluated by designing four different downstream purification schemes, where the use of affinity chromatography (protein A and L) was the most efficient to isolate the Fab fragments after IgG digestion. A rapid screening of optimal conditions for the binding of pure Fab fragments to four non-affinity chromatography resins was then performed using micro-columns fabricated in a PDMS microfluidic device. The goal of these studies was to screen and evaluate the performance of two cation exchange (carboxymethyl and heparin) and two multimodal (Capto MMC and phenyl boronate) ligands for the capture of Fabs under a wide range of pH (5–9) and conductivity (up to 8 mS/cm) conditions. Multimodal resins showed the best results in binding Fab fragments, particularly at pH 5, well below the range of isoelectric points of the target Fab molecules. In addition, these resins demonstrated to have a salt-tolerant behaviour, meaning that the binding of Fab fragments was not significantly impacted when the conductivity of the adsorption buffer was increased to near-physiological conditions (8 mS/cm).

Single-step purification of cyclotides using affinity chromatography.

Cyclotides are considered promising scaffolds for drug development owing to their inherent host defence activities and highly stable structure, defined by the cyclic cystine knot. These proteins are expressed as complex mixtures in plants. Although several methods have been developed for their isolation and analysis, purification of cyclotides is still a lengthy process. Here, we describe the use of affinity chromatography for the purification of cyclotides using polyclonal IgG antibodies raised in rabbits against cycloviolacin O2 and immobilized on NHS-activated Sepharose columns. Cycloviolacin O2 was used as a model substance to evaluate the chromatographic principle, first as a pure compound and then in combination with other cyclotides, that is, bracelet cyclotide cycloviolacin O19 and Möbius cyclotide kalata B1, and in a plant extract. We demonstrate that single-step purification of cyclotides by affinity chromatography is possible but cross reactivity may occur between homologue cyclotides of the bracelet subfamily.

Comparative binding to DR4 and DR5 receptors of TRAIL and BNNTs/PAHE/mPEG-DSPE/TRAIL nanoparticles.

TRAIL is a member of the tumor necrosis factor family of cytokines, which induces apoptosis of cancer cells, thanks to its binding to its cognate receptors DR5 and DR4. We have recently demonstrated that nanovectorization of TRAIL with single-walled carbon nanotubes enhanced TRAIL affinity to DR5. In this paper, 1-pyrenebutyric acid N-hydroxysuccinimide ester functionalized boron nitride nanotubes (BNNTs) were used to anchor the TRAIL protein. The resulting BNNT/1-pyrenebutyric acid N-hydroxysuccinimide ester nanotubes were mixed with methoxy-poly(ethylene glycol)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-conjugates so as to allow a good dispersion of these nanoparticle TRAIL (NPT) in aqueous solution. The difference of binding between NPT and soluble TRAIL to DR4 and DR5 receptors was then studied by the use of affinity chromatography. DR4 and DR5 receptors were thus immobilized on a chromatographic support, and the binding of the 2 ligands TRAIL and NPT to DR4 and DR5 was studied in the temperature range 30°C to 50°C. Negative enthalpy (ΔH) values indicated that van der Waals interactions and hydrogen bonding are engaged favorably at the ligand-receptor interface. It was shown that their rank-ordered affinities were strongly different in the sequence TRAILDR4 <NPTDR4 <TRAILDR5 <NPTDR5 , and the highest affinity for NPT to DR4 and DR5 receptors observed at low pHs was due to the less accessibility of the His molecular switch to be protonated when TRAIL was immobilized on BNNTs. Taken together, our results demonstrated that nanovectorization of TRAIL with BNNTs enhanced its binding to both DR4 and DR5 receptors at 37°C. Our novel nanovector could potentially be used for delivering TRAIL to cells for cancer treatment.

Removal of protein S1 from Escherichia coli ribosomes without the use of affinity chromatography

The paper reports an inexpensive and efficient procedure for the removal of protein S1 from E. coli ribosomes. It comprises incubation of ribosomes in a pyrimidine polyribonucleotide solution followed by centrifugation of the sample through a sucrose cushion. To avoid co-sedimentation of the S1-bound polypyrimidine with the ribosomes, its length should not exceed several hundred nucleotides. Unlike popular affinity chromatography through a poly(U) Sepharose or poly(U) cellulose column, the method tolerates limited polyribonucleotide degradation by eventual traces of ribonucleases, and can readily be incorporated into standard protocols for the isolation of ribosomes by centrifugation.

Crystallization and X-ray diffraction analysis of an L-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii and a D-xylonate dehydratase from Caulobacter crescentus.

L-Arabinonate dehydratase (EC 4.2.1.25) and D-xylonate dehydratase (EC 4.2.1.82) are two enzymes that are involved in a nonphosphorylative oxidation pathway of pentose sugars. L-Arabinonate dehydratase converts L-arabinonate into 2-dehydro-3-deoxy-L-arabinonate, and D-xylonate dehydratase catalyzes the dehydration of D-xylonate to 2-dehydro-3-deoxy-D-xylonate. L-Arabinonate and D-xylonate dehydratases belong to the IlvD/EDD family, together with 6-phosphogluconate dehydratases and dihydroxyacid dehydratases. No crystal structure of any L-arabinonate or D-xylonate dehydratase is available in the PDB. In this study, recombinant L-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii (RlArDHT) and D-xylonate dehydratase from Caulobacter crescentus (CcXyDHT) were heterologously expressed in Escherichia coli and purified by the use of affinity chromatography followed by gel-filtration chromatography. The purified proteins were crystallized using the hanging-drop vapour-diffusion method at 293 K. Crystals of RlArDHT that diffracted to 2.40 Å resolution were obtained using sodium formate as a precipitating agent. They belonged to space group P21, with unit-cell parameters a=106.07, b = 208.61, c = 147.09 Å, β = 90.43°. Eight RlArDHT molecules (two tetramers) in the asymmetric unit give a VM value of 3.2 Å(3) Da(-1) and a solvent content of 62%. Crystals of CcXyDHT that diffracted to 2.66 Å resolution were obtained using sodium formate and polyethylene glycol 3350. They belonged to space group C2, with unit-cell parameters a = 270.42, b = 236.13, c=65.17 Å, β=97.38°. Four CcXyDHT molecules (a tetramer) in the asymmetric unit give a VM value of 4.0 Å(3) Da(-1) and a solvent content of 69%.

Optimization of pore structure and particle morphology of mesoporous silica for antibody adsorption for use in affinity chromatography

Antibodies have received significant attention for use as antibody drugs, because they bind the objective protein (antigen) via antigen–antibody reactions. Recently, many reports have appeared on various monoclonal antibodies that recognize a single antigen. In this study, monoclonal antibodies are used as adsorbates on mesoporous silica (MPS) for affinity chromatography. MPS has high surface area and large pore volume; moreover, pore diameter, pore structure, and particle morphology are relatively easy to tune by adjusting the conditions of synthesis. The pore structure (two-dimensional (2D) hexagonal and three-dimensional cubic) and particle morphology (spherical and polyhedral) of MPS are optimized for use in a monoclonal antibody/MPS composite. When anti-IgG (one of the monoclonal antibodies) adsorbs on the MPS material and IgG (antigen) binds to anti-IgG/MPS composites, MCM-41p with a 2D-hexagonal pore structure and polyhedral particle morphology has the highest IgG binding efficiency. In addition, the antibody/MPS composites remain stable in chaotropic and low-pH solutions and can be cycled at least five times without decreasing IgG elution. In purification and removal tests, the use of the antibody/MPS composites allows only the objective protein from protein mixtures to be bound and eluted.

Dataset concerning GroEL chaperonin interaction with proteins.

GroEL chaperonin is well-known to interact with a wide variety of polypeptide chains. Here we show the data related to our previous work (http://dx.doi.org/10.1016/j.pep.2015.11.020[1]), and concerning the interaction of GroEL with native (lysozyme, α-lactalbumin) and denatured (lysozyme, α-lactalbumin and pepsin) proteins in solution. The use of affinity chromatography on the base of denatured pepsin for GroEL purification from fluorescent impurities is represented as well.

Reconstitution of Fungal Nonribosomal Peptide Synthetases in Yeast and In Vitro.

The emergence of next-generation sequencing has provided new opportunities in the discovery of new nonribosomal peptides (NRPs) and NRP synthethases (NRPSs). However, there remain challenges for the characterization of these megasynthases. While genetic methods in native hosts are critical in elucidation of the function of fungal NRPS, in vitro assays of intact heterologously expressed proteins provide deeper mechanistic insights in NRPS enzymology. Our previous work in the study of NRPS takes advantage of Saccharomyces cerevisiae strain BJ5464-npgA as a robust and versatile platform for characterization of fungal NRPSs. Here we describe the use of yeast recombination strategies in S. cerevisiae for cloning of the NRPS coding sequence in 2μ-based expression vector; the use of affinity chromatography for purification of NRPS from the total S. cerevisiae soluble protein fraction; and strategies for reconstitution of NRPSs activities in vitro.

Affinity Chromatography of Native and Recombinant Proteins from Receptors for Insulin and IGF-I to Recombinant Single Chain Antibodies.

Affinity chromatography is an efficient method to isolate proteins by taking advantage of their affinities for specific molecules such as substrates, inhibitors, antigens, ligands, antibodies, and other interacting molecules, including subunits. Nowadays, we take the effectiveness and excellence of this technology for granted. This essay will mainly cover the use of affinity chromatography based on my experience.

Fibronectin-, vitronectin- and laminin-binding proteins at the cell walls of Candida parapsilosis and Candida tropicalis pathogenic yeasts.

BackgroundCandida parapsilosis and C. tropicalis increasingly compete with C. albicans—the most common fungal pathogen in humans—as causative agents of severe candidiasis in immunocompromised patients. In contrast to C. albicans, the pathogenic mechanisms of these two non-albicans Candida species are poorly understood. Adhesion of Candida yeast to host cells and the extracellular matrix is critical for fungal invasion of hosts.MethodsThe fungal proteins involved in interactions with extracellular matrix proteins were isolated from mixtures of β-1,3-glucanase– or β-1,6-glucanase–extractable cell wall-associated proteins by use of affinity chromatography and chemical cross-linking methods, and were further identified by liquid chromatography-coupled tandem mass spectrometry.ResultsIn the present study, we characterized the binding of three major extracellular matrix proteins—fibronectin, vitronectin and laminin—to C. parapsilosis and C. tropicalis pseudohyphae. The major individual compounds of the fungal cell wall that bound fibronectin, vitronectin and laminin were found to comprise two groups: (1) true cell wall components similar to C. albicans adhesins from the Als, Hwp and Iff/Hyr families; and (2) atypical (cytoplasm-derived) surface-exposed proteins, including malate synthase, glucose-6-phosphate isomerase, 6-phosphogluconate dehydrogenase, enolase, fructose-1,6-bisphosphatase, transketolase, transaldolase and elongation factor 2.DiscussionThe adhesive abilities of two investigated non-albicans Candida species toward extracellular matrix proteins were comparable to those of C. albicans suggesting an important role of this particular virulence attribute in the pathogenesis of infections caused by C. tropicalis and C. parapsilosis.ConclusionsOur results reveal new insight into host–pathogen interactions during infections by two important, recently emerging, fungal pathogens.

Protein purification using PDZ affinity chromatography.

PDZ domains function in nature as protein-binding domains within scaffold and membrane-associated proteins. They comprise approximately 90 residues and undergo specific, high-affinity interactions with complementary C-terminal peptide sequences, other PDZ domains, and/or phospholipids. We have previously shown that the specific, strong interactions of PDZ domains with their ligands make them well suited for use in affinity chromatography. This unit provides protocols for the PDZ affinity chromatography procedure that are applicable for the purification of proteins that contain PDZ domains or PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We detail the preparation of affinity resins composed of PDZ domains or PDZ domain peptide ligands coupled to solid supports. These resins can be used to purify proteins containing endogenous or genetically introduced PDZ domains or ligands, eluting the proteins with free PDZ domain peptide ligands.

Expression of G protein-coupled receptors in Mammalian cells.

G protein-coupled receptors (GPCRs) are cell surface proteins and play crucial role in mediating effective communication between extracellular and intracellular milieu of the cell. To understand the structure and function of these membrane proteins, it is imperative to express the proteins in a functional form and in sufficient quantities. However, heterologous expression of GPCRs in sufficient amounts for structural studies is a daunting task and over the years researchers have tried various expression systems to achieve this goal. Here, we describe a method developed in the laboratory of Dr. Har Gobind Khorana and successfully used over the past decade to express GPCRs and other membrane proteins in mammalian cells. Improvements to this method include use of genes codon optimized for expression in mammalian cells, use of expression tags at N-terminus, and affinity tags at the N-terminus and C-terminus of GPCRs. It also provides details for the process of selection for stably transfected clones, and to identify clones that are effectively expressing the GPCR of interest on the cell surface using flow cytometry. Purification of the overexpressed GPCR involves use of affinity chromatography. The method discussed here can also be effectively used to express cytotoxic proteins such as some constitutively active GPCRs. The method is robust and reliable to express GPCRs.

Engineering of novel Staphylococcal Protein A ligands to enable milder elution pH and high dynamic binding capacity

We describe novel Staphylococcal Protein A ligands that enable milder elution pH for use in affinity chromatography. The change in elution pH is the result of point mutations to the protein sequence. Two novel ligands are investigated in this study. The first, designated Z(H18S)4, represents a histidine to serine substitution single mutation. The second, designated Z(H18S, N28A)4, is a double mutant comprising histidine to serine and asparagine to alanine mutations. Both are compared against the unmutated sequence, designated Z4, which is currently utilized in a commercially available Protein A stationary phase for the purification of molecules containing Fc domains. The ligands are coupled to a chromatography support matrix and tested against a panel of antibodies and an Fc fusion protein for elution pH, dynamic binding capacity, step-wise elution, and capture from clarified culture media. Results demonstrate that the novel ligands result in milder elution pH, on average >0.5 pH units, when tested in a pH gradient. For step-wise elution at pH 4.0, the Z(H18S, N28A)4 ligand showed on average a greater than 30% increase in yield compared to Z4. Importantly, for the antibodies tested the mutations did not result in a decrease in dynamic binding capacity or other desirable attributes such as selectivity. A potential application of the novel ligands is shown with a pH sensitive molecule prone to aggregation under acidic conditions.

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

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.

PDZ affinity chromatography: A general method for affinity purification of proteins based on PDZ domains and their ligands

PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ∼90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and β-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins.