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A. S. Kamalanathan

Bio: A. S. Kamalanathan is an academic researcher from VIT University. The author has contributed to research in topic(s): Affinity chromatography & Glycan. The author has an hindex of 7, co-authored 14 publication(s) receiving 97 citation(s).
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Journal ArticleDOI
TL;DR: Fast flow metal chelate methacrylate monolithic system - CIM (Convective Interaction Media) disk chelated with Cu(II) disk was found to be highly efficient in capturing His-containing peptides with high degree of specificity and selectivity and demonstrated a significant reduction in sample complexity.
Abstract: In recent years, bottom-up approach has become the popular method of choice for large scale analysis of complex proteome samples. Peptide fractionation determines the efficiency of the bottom-up method and often the resolving power of reverse phase liquid chromatography (RPLC) is insufficient for efficient protein identification in case of complex biological samples. To overcome the inherent limitation of proteomics associated with sample complexity, we evaluated fast flow metal chelate methacrylate monolithic system – CIM (Convective Interaction Media) disk chelated with Cu(II) for targeted affinity selection of histidine-containing peptides. Initially the Cu(II)-IMAC using CIM disk was evaluated using tryptic digest of protein mixtures of 8 model proteins and was found to be highly efficient in capturing His-containing peptides with high degree of specificity and selectivity. Further the efficiency of His-peptide enrichment using CIM-IMAC was also demonstrated using complex biological samples like total Escherichia coli cell lysate. The analysis of the Cu(II)-IMAC retained peptides from tryptic digests of model protein mixture and E. coli not only demonstrated a significant reduction in sample complexity but also subsequently enabled the identification of additional peptides. His-peptide enrichment also enabled the identification of low abundant proteins that were not detected in the analysis of total E. coli digest.

20 citations


Journal ArticleDOI
TL;DR: This new monolithic CIM-disk system with L-histidine immobilized: immobilized histidine affinity chromatography (IHAC) constitutes a good tool allowing the fast and selective purification of bioactive oligouronides.
Abstract: Selective purification of alpha-(1,4)-oligogalacturonides was investigated using several pseudobioaffinity chromatography matrix with aminoacid L-histidine as pseudobiospecific ligand: (1) sepharose 4B-bisoxyran-histidine, (2) sepharose 4B-epoxy-histidine, (3) silica-oxyran-histidine and (4) CIM-disk-EDA-histidine. These anionic oligosaccharides prepared by enzymatic and chemical cleavage of polygalacturonic acid were used as models sugar in order to optimize the adsorption and elution parameters for a selective purification of bioactive oligouronides. Monolithic CIM-disk chromatography is one of the fastest liquid chromatographic method using for separation and purification of biomolecules thanks to high mass transfer rate. In this way, this new monolithic CIM-disk system with L-histidine immobilized: immobilized histidine affinity chromatography (IHAC) constitutes a good tool allowing the fast and selective purification of bioactive oligouronides.

14 citations


Journal ArticleDOI
TL;DR: The results indicate the high potential of this method for purification of total IgG from complex biological sources and also for separation of IgG1 from other subclasses.
Abstract: The pseudobiospecific affinity ligand l-histidine was immobilized on epoxy, carbonyldiimidazole (CDI), and ethylenediamine (EDA) convective interaction media (BIA Separations, Slovenia) monolithic disks to obtain the histidyl affinity column for purification of immunoglobulin G (IgG) The kinetics and the mass transfer properties of the affinity columns were studied to determine the optimum buffer condition, flow rate, and concentration of IgG for maximum IgG adsorption The binding capacities of all the three affinity columns were higher with zwitterionic buffer morpholinopropanesulfonic acid than with charged buffers such as tris-HCl and phosphate buffers, and the optimum pH was 65 The interaction of IgG with histidine immobilized CDI and epoxy disks was found to be predominantly driven by ionic interaction, while the interaction with EDA-histidine disk could be partially governed by multiple non-covalent forces of interaction The maximum binding capacity (Qm ) of l-histidine immobilized on EDA-, CDI-, and epoxy-activated convective interaction media disks were 1983 ± 025, 1585 ± 018 and 1211 ± 017 mg/ml of support, respectively, and the dissociation constant (Kd ) were calculated to be in the micromolar range for all the three histidyl monolithic columns Purification of IgG from untreated human serum was also attempted, and the results indicate the high potential of this method for purification of total IgG from complex biological sources and also for separation of IgG1 from other subclasses

13 citations


Journal ArticleDOI
TL;DR: The results of the studies suggested that the inhibition of the DPP-IV enzyme as one of the pathways by which the Aloe vera extract may restore the pancreatic islets cell mass in diabetic animal model.
Abstract: Aloe vera, a succulent herb, has a long history of use in traditional medicine, including diabetes. Earlier studies from our laboratory demonstrated that the Aloe vera extract has the ability to inhibit the diabetic drug target dipeptidyl peptidase (DPP) IV in vitro. This current study focuses on the isolation of small water soluble active molecule(s) involved in DPP-IV inhibition from Aloe vera extract, and further to characterize its structure and to elucidate the mode of inhibition of the DPP-IV enzyme. Aloe vera gel ethanolic extract was subjected to preparative reverse-phase high-pressure liquid chromatography (RP-HPLC), LH-20 Sephadex gel filtration chromatography, followed by analytical RP-HPLC, to isolate the active molecule involved in DPP-IV inhibition. Based on the spectroscopic studies, the structure of the isolated DPP-IV inhibitor was predicted to be 3, 6-dioxo-3, 3a, 6, 6 a-tetrahydropyrrolo [3, 4-c] pyrrole-1, 4-dicarboxamide with the chemical formula C8H6N4O4, having the molecular weight of 225.175 Da. This molecule inhibited the DPP-IV enzyme in a noncompetitive manner with an IC50 value of 8.59 ± 2.61 µM, with a Ki of 4.7 ± 0.038 µM. Thus, the mechanism of DPP-IV inhibition and the inhibitory constants were determined. The results of our studies suggested that the inhibition of the DPP-IV enzyme as one of the pathways by which the Aloe vera extract may restore the pancreatic islets cell mass in diabetic animal model.

9 citations


Journal ArticleDOI
TL;DR: Glycan array analysis of DBL revealed its affinity toward high mannose N-linked glycans with enhanced affinity for terminalMannose including N- linked glycans of HIV envelope glycoprotein gp120 and has strong anti-reverse transcriptase activity.
Abstract: Dioscorea bulbifera or air potato has been used as a folk remedy to treat cancer. A mannose binding lectin from bulbils of D. bulbifera was purified in a single step by affinity chromatography on mucin coupled Sepharose 4B column, determined by its fine sugar specificity by glycan array analysis and studied for its clinical potential in cancer and HIV research. SDS-PAGE showed that lectin is a monomer of Mr 24kDa. DBL agglutinated only rabbit erythrocytes and was inhibited by mucin, asialomucin, fetuin, asialofetuin and transferrin but not by any monosaccharides. Glycan array analysis of DBL revealed its affinity toward high mannose N-linked glycans with enhanced affinity for terminal mannose including N-linked glycans of HIV envelope glycoprotein gp120 and has strong anti-reverse transcriptase activity. DBL showed strong binding to non-metastatic human colon epithelial cancer HT 29, metastatic SW 620 and hepatocellular HepG2 cell lines. DBL showed dose and time dependent growth inhibitory effects on all the three cell lines HT 29, SW 620 and HepG2 with IC50 of 110μg, 9.8μg, 40μg respectively at 72h. Inhibitory effect of DBL was effectively blocked in presence of competing glycans like mucin. DBL has promising clinical potential both in cancer and HIV research.

9 citations


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Journal ArticleDOI
Nika Kruljec1, Tomaž Bratkovič1Institutions (1)
TL;DR: The design, development, and properties of diverse classes of alternative antibody-binding ligands, ranging from engineered versions of Ig-binding proteins, to artificial binding proteins, peptides, aptamers, and synthetic small-molecular-weight compounds are reviewed.
Abstract: The demand for recombinant therapeutic antibodies and Fc-fusion proteins is expected to increase in the years to come. Hence, extensive efforts are concentrated on improving the downstream processing. In particular, the development of better-affinity chromatography matrices, supporting robust time- and cost-effective antibody purification, is warranted. With the advances in molecular design and high-throughput screening approaches from chemical and biological combinatorial libraries, novel affinity ligands representing alternatives to bacterial immunoglobulin (Ig)-binding proteins have entered the scene. Here, we review the design, development, and properties of diverse classes of alternative antibody-binding ligands, ranging from engineered versions of Ig-binding proteins, to artificial binding proteins, peptides, aptamers, and synthetic small-molecular-weight compounds. We also provide examples of applications for the novel affinity matrices in chromatography and beyond.

48 citations


Journal ArticleDOI
01 Mar 2017-Methods
TL;DR: A detailed overview on affinity chromatography and the components involved in purification is provided to provide a detailed overview of antibody purification methodologies and their underlying working principles.
Abstract: Antibodies continue to be extremely utilized entities in myriad applications including basic research, imaging, targeted delivery, chromatography, diagnostics, and therapeutics. At production stage, antibodies are generally present in complex matrices and most of their intended applications necessitate purification. Antibody purification has always been a major bottleneck in downstream processing of antibodies, due to the need of high quality products and associated high costs. Over the years, extensive research has focused on finding better purification methodologies to overcome this holdup. Among a plethora of different techniques, affinity chromatography is one of the most selective, rapid and easy method for antibody purification. This review aims to provide a detailed overview on affinity chromatography and the components involved in purification. An array of support matrices along with various classes of affinity ligands detailing their underlying working principles, together with the advantages and limitations of each system in purifying different types of antibodies, accompanying recent developments and important practical methodological considerations to optimize purification procedure are discussed.

40 citations


Journal ArticleDOI
Ana C. A. Sousa1, D. Bicho1, Cândida T. Tomaz1, Fani Sousa1  +1 moreInstitutions (1)
TL;DR: The binding capacity has been found to be significantly higher for sc plasmid, probably because of its compact structure, being also improved when using feedstock with increased plasmids concentrations and decreased linear velocity.
Abstract: The use of therapeutics based on plasmid DNA (pDNA) relies on procedures that efficiently produce and purify the supercoiled (sc) plasmid isoform. Several chromatographic methods have been applied for the sc plasmid purification, but with most of them it is not possible to obtain the required purity degree and the majority of the supports used present low capacity to bind the plasmid molecules. However, the chromatographic monolithic supports are an interesting alternative to conventional supports due to their excellent mass transfer properties and their high binding capacity for pDNA. The separation of pDNA isoforms, using short non-grafted monolithic column with CarbonylDiImidazole (CDI) functional groups, is described in the current work. The effect of different flow rates on plasmid isoforms separation was also verified. Several breakthrough experiments were designed to study the effect of different parameters such as pDNA topology and concentration as well as flow rate on the monolithic support binding capacity. One of the most striking results is related to the specific recognition of the sc isoform by this CDI monolith, without flow rate dependence. Additionally, the binding capacity has been found to be significantly higher for sc plasmid, probably because of its compact structure, being also improved when using feedstock with increased plasmid concentrations and decreased linear velocity. In fact, this new monolithic support arises as a powerful instrument on the sc pDNA purification for further clinical applications.

40 citations


Journal ArticleDOI
TL;DR: This mini-review reports the contributions of several groups to the development of macroporous monoliths and their modification by immobilization of specific ligands on the products for their application in affinity chromatography.
Abstract: In the early 1990s, three research groups simultaneously developed continuous macroporous rod-shaped polymeric systems to eliminate the problem of flow through the interparticle spaces generally presented by the chromatography columns that use particles as filler. The great advantage of those materials, forming a continuous phase rod, is to increase the mass transfer by convective transport, as the mobile phase is forced to go through all means of separation, in contrast to particulate media where the mobile phase flows through the interparticle spaces. Due to their special characteristics, the monolithic polymers are used as base-supports in different separation techniques, those chromatographic processes being the most important and, to a greater extent, those involving the separation of biomolecules as in the case of affinity chromatography. This mini-review reports the contributions of several groups to the development of macroporous monoliths and their modification by immobilization of specific ligands on the products for their application in affinity chromatography.

37 citations


Journal ArticleDOI
TL;DR: One of the most striking results shows that histidine interacts preferentially with guanine, and the presence of secondary structures on polyA and polyG oligonucleotides has a significant influence on retention.
Abstract: The recent application of histidine-agarose affinity supports in plasmid purification takes advantage of the biorecognition of nucleic acid bases by the histidine ligand. This consideration prompted the need for better understanding the interactions involved in affinity chromatography of plasmid DNA with the histidine-agarose support. In this work, we used synthetic homo-deoxyoligonucleotides with different sizes (1-30 nucleotides long), to explore the effect of several conditions like hydrophobic character of the individual bases, presence of secondary structures, temperature, pH and salt concentration on the mechanism of retention of nucleic acids to histidine-agarose support. One of the most striking results shows that histidine interacts preferentially with guanine, and the presence of secondary structures on polyA and polyG oligonucleotides has a significant influence on retention. Otherwise, the temperature manipulation has not shown a direct influence on oligonucleotide retention, only inducing conformational changes on secondary structures. Overall, the results obtained provide valuable information for the future development and implementation of histidine and other amino acids as ligands in chromatography for the purification of plasmid DNA and other nucleic acids, by improving the knowledge of the interactions involved as well as of the parameters influencing the retention.

32 citations


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Author's H-index: 7

No. of papers from the Author in previous years
YearPapers
20201
20192
20183
20172
20161
20151

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Author's top 5 most impactful journals

Journal of Chromatography B

4 papers, 42 citations

Journal of Molecular Recognition

3 papers, 27 citations

Journal of Chromatography A

1 papers, 1 citations