Showing papers on "Affinity chromatography published in 2019"
TL;DR: This mini review summarizes and describes the advances, results, and impact on the Protein A chromatography purification processing.
Abstract: Antibodies for therapeutic use are being continuously approved and their demand has been steadily growing. As known, the golden standard for monoclonal antibody (mAb) purification is Protein A affinity chromatography, a technology that has gained high interest because of its great performance and capabilities. The main concerns are the elevated resins costs and their limited lifetime compared to other resins (e.g. ion exchange chromatography). Great efforts have been carried out to improve purification conditions, such as resin characterization and designing alkali/acid stable resins with a longer lifetime. Modification of Protein A ligands and alternative formats such as monoliths membranes and microshperes have been tested to increase the purification performance. New technology has been proposed to improve the large-scale separation; in addition, alternative ligands have been suggested to capture mAbs instead of Protein A ligand; however, most of the information is locked by pharmaceutical companies. This mini review summarizes and describes the advances, results, and impact on the Protein A chromatography purification processing.
72 citations
TL;DR: The developed method is easily scalable to pilot and process scale and allows a fast accomplishment of this separation within one day, in contradiction to other protocols where the extracellular vesicles are recovered by binding to heparin affinity chromatography.
58 citations
TL;DR: The protein binding rates of structurally different flavonoids to human serum albumin (HSA) were elucidated by applying the high performance affinity chromatography (HPAC) and it was found that an additional methoxy group in flavone ring A would decrease the protein binding rate.
55 citations
TL;DR: Results indicate that bioactive SΔTM was expressed in silkworm larvae and S protein-displaying nanovesicles from Bm5 cells may lead to the development of nanoparticle-based vaccines against MERS-CoV and the diagnostic detection of MSPV.
52 citations
TL;DR: This work describes the development of MIPs, based on the epitope approach, synthesized from the tetrapeptide DYKD as template that affords purification of FLAG-derived recombinant proteins.
Abstract: Epitope tagging is widely used to fuse a known epitope to proteins for which no affinity receptor is available by using recombinant DNA technology. One example is FLAG epitope (DYKDDDDK), which provides better purity and recoveries than the favorite poly histidine tag. However, purification requires using anti-FLAG antibody resins, the high cost and nonreusability of which restrict widespread use. One cost-effective solution is provided by the use of bioinspired anti-FLAG molecularly imprinted polymers (MIPs). This work describes the development of MIPs, based on the epitope approach, synthesized from the tetrapeptide DYKD as template that affords purification of FLAG-derived recombinant proteins. Polymer was optimized by using a combinatorial approach to select the functional monomer(s) and cross-linker(s), resulting in the best specific affinity toward FLAG and the peptide DYKD. The imprinted resin obtained was used to purify mCherry proteins tagged with either FLAG or DYKD epitopes from crude cell lysa...
42 citations
TL;DR: A lectin affinity purification-mass spectrometry approach to characterize lectin-reactive glycoproteoforms and elucidate lectin specificities at the intact protein level is presented.
Abstract: Lectins are carbohydrate binding proteins that recognize specific epitopes present on target glycoproteins. Changes in lectin-reactive carbohydrate repertoires are related to many biological signaling pathways and recognized as hallmarks of several pathological processes. Consequently, lectins are valuable probes, commonly used for examining glycoprotein structural and functional microheterogeneity. However, the molecular interactions between a given lectin and its preferred glycoproteoforms are largely unknown due to the inherent complexity and limitations of methods used to investigate intact glycoproteins. Here, we apply a lectin-affinity purification procedure coupled with native mass spectrometry to characterize lectin-reactive glycoproteoforms at the intact protein level. We investigate the interactions between the highly fucosylated and highly branched glycoproteoforms of haptoglobin and α1-acid glycoprotein using two different lectins Aleuria aurantia lectin (AAL) and Phaseolus vulgaris leucoagglutinin (PHA-L), respectively. Firstly we show a co-occurrence of fucosylation and N-glycan branching on haptoglobin, particularly among highly fucosylated glycoproteoforms. Secondly, we analyze the global heterogeneity of highly branched glycoproteoforms of haptoglobin and α1-acid glycoprotein and reveal that while multi-fucosylation attenuates the lectin PHA-L binding to haptoglobin, it has no impact on AGP. Taken together, our lectin affinity purification native MS approach elucidates lectin specificities between intact glycoproteins, not achievable by other methods. Moreover, since aberrant glycosylation of Hp and AGP are potential markers for many diseases, including pancreatic, hepatic and ovarian cancers, understanding their interactions with lectins will help the development of carbohydrate-centric monitoring methods to understand their pathophysiological implications.
42 citations
TL;DR: The nanoMIPs prepared for Fc domain and epitope demonstrated a specific recognition of both human and goat IgGs, therefore they could be considered as a synthetic analogue of protein A and benefit from its intrinsic stability, short time and low cost of preparation.
37 citations
TL;DR: This work hyphenated FcɣRIIIa affinity chromatography to mass spectrometry, which allowed direct affinity comparison of glycoforms of intact monoclonal antibodies, and greatly improves the understanding of individual glycoform structure–function relationships.
Abstract: Determination of the impact of individual antibody glycoforms on FcɣRIIIa affinity, and consequently antibody-dependent cell-mediated cytotoxicity (ADCC) previously required high purity glycoengineering. We hyphenated FcɣRIIIa affinity chromatography to mass spectrometry, which allowed direct affinity comparison of glycoforms of intact monoclonal antibodies. The approach enabled reproduction and refinement of known glycosylation effects, and insights on afucosylation pairing as well as on low-abundant, unstudied glycoforms. Our method greatly improves the understanding of individual glycoform structure-function relationships. Thus, it is highly relevant for assessing Fc-glycosylation critical quality attributes related to ADCC.
34 citations
20 Sep 2019
TL;DR: This review aims to discuss affinity purification technologies and address the principles, advantages, limitations and potential applications of them.
Abstract: Recombinant proteins have wide applications in the development of pharmaceutical compounds, industrial applications of enzymes, and basic proteomics research. In this way, efficient production of r...
30 citations
TL;DR: The GMGL gene was expressed in Escherichia coli as a His-tag fusion protein and purified by affinity chromatography with a yield of 264 mg per liter fermentation broth, and showed the highest hydrolysis activity at 60 °C and pH 8.0, and the half-life was 60 min at 70 °C.
Abstract: Lipases with unique substrate specificity are highly desired in biotechnological applications. In this study, a putative marine Geobacillus sp. monoacylglycerol lipase (GMGL) encoded gene was identified by a genomic mining strategy. The gene was expressed in Escherichia coli as a His-tag fusion protein and purified by affinity chromatography with a yield of 264 mg per liter fermentation broth. The recombinant GMGL shows the highest hydrolysis activity at 60 °C and pH 8.0, and the half-life was 60 min at 70 °C. The GMGL is active on monoacylglycerol (MAG) substrate but not diacylglycerol (DAG) or triacylglycerol (TAG), and produces MAG as the single product in the esterification reaction. Modeling structure analysis showed that the catalytic triad is formed by Ser97, Asp196 and His226, and the flexible cap region is constituted by residues from Ala120 to Thr160. A mutagenesis study on Leu142, Ile145 and Ile170 located in the substrate binding tunnel revealed that these residues were related with its substrate specificity. The kcat/Km value toward the pNP-C6 substrate in mutants Leu142Ala, Ile145Ala and Ile170Phe increased to 2.3-, 1.4- and 2.2-fold as compared to that of the wild type, respectively.
28 citations
TL;DR: Study on IgG separation from human serum showed that the hybrid biomimetic ligand could provide comparable IgG purity and recovery, but much milder elution condition as those of Protein A affinity chromatography.
TL;DR: This α-amylase gene from Pseudoalteromonas sp.
TL;DR: The data demonstrates that copurification of Cathepsin L on nonaffinity modalities is principally because of similar retention on the stationary phase and not through interactions with product, and exhibits a broad elution profile in cation exchange chromatography (CEX) likely because of its different forms.
Abstract: A stochastic approach of copurification of the protease Cathepsin L that results in product fragmentation during purification processing and storage is presented. Cathepsin L was identified using mass spectroscopy, characterization of proteolytic activity, and comparison with fragmentation patterns observed using recombinant Cathepsin L. Cathepsin L existed in Chinese hamster ovary cell culture fluids obtained from cell lines expressing different products and cleaved a variety of recombinant proteins including monoclonal antibodies, antibody fragments, bispecific antibodies, and fusion proteins. Therefore, characterization its chromatographic behavior is essential to ensure robust manufacturing and sufficient shelf life. The chromatographic behaviors of Cathepsin L using a variety of techniques including affinity, cation exchange, anion exchange, and mixed mode chromatography were systematically evaluated. Our data demonstrates that copurification of Cathepsin L on nonaffinity modalities is principally because of similar retention on the stationary phase and not through interactions with product. Lastly, Cathespin L exhibits a broad elution profile in cation exchange chromatography (CEX) likely because of its different forms. Affinity purification is free of fragmentation issue, making affinity capture the best mitigation of Cathepsin L. When affinity purification is not feasible, a high pH wash on CEX can effectively remove Cathepsin L but resulted in significant product loss, while anion exchange chromatography operated in flow-through mode does not efficiently remove Cathepsin L. Mixed mode chromatography, using Capto™ adhere in this example, provides robust clearance over wide process parameter range (pH 7.7 ± 0.3 and 100 ± 50 mM NaCl), making it an ideal technique to clear Cathepsin L. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2732, 2019.
TL;DR: The use of solid supported proteins has significantly contributed to the development of automated and reliable screening methods that enable ligands to be isolated and characterized in complex matrixes without purification, thereby reducing costs and avoiding time-laborious steps.
Abstract: Ligand-target interactions play a central role in drug discovery processes because these interactions are crucial in biological systems. Small molecules-proteins interactions can regulate and modulate protein function and activity through conformational changes. Therefore, bioanalytical tools to screen new ligands have focused mainly on probing ligand-target interactions. These interactions have been evaluated by using solid-supported proteins, which provide advantages like increased protein stability and easier protein extraction from the reaction medium, which enables protein reuse. In some specific approaches, precisely in the ligand fishing assay, the bioanalytical method allows the ligands to be directly isolated from complex mixtures, including combinatorial libraries and natural products extracts without prior purification or fractionation steps. Most of these screening assays are based on liquid chromatography separation, and the binding events can be monitored through on-line or off-line methods. In the on-line approaches, solid supports containing the immobilized biological target are used as chromatographic columns most of the time. Several terms have been used to refer to such approaches, such as weak affinity chromatography, high-performance affinity chromatography, on-flow activity assays, and high-performance liquid affinity chromatography. On the other hand, in the off-line approaches, the binding event occurs outside the liquid chromatography system and may encompass affinity and activity-based assays in which the biological target is immobilized on magnetic particles or monolithic silica, among others. After the incubation step, the supernatant or the eluate from the binding assay is analyzed by liquid chromatography coupled to various detectors. Regardless of the selected bioanalytical approach, the use of solid supported proteins has significantly contributed to the development of automated and reliable screening methods that enable ligands to be isolated and characterized in complex matrixes without purification, thereby reducing costs and avoiding time-laborious steps. This review provides a critical overview of recently developed assays.
TL;DR: The recombinant chitinase from C. violaceum ATCC 12472 inhibited the conidia germination of the phytopathogenic fungi Fusarium oxysporum and F. guttiforme, hence preventing mycelial growth and showing the potential of CvChi47 as a molecular tool to control plant diseases caused by these Fus aquarium species.
TL;DR: Two PLA2-CB isoforms in a prokaryotic system showed phospholipase activity and virucidal effects against chikungunya virus, dengue virus, yellow fever virus and Zika virus.
Abstract: The global emergence and re-emergence of arthropod-borne viruses (arboviruses) over the past four decades have become a public health crisis of international concern, especially in tropical and subtropical countries. A limited number of vaccines against arboviruses are available for use in humans; therefore, there is an urgent need to develop antiviral compounds. Snake venoms are rich sources of bioactive compounds with potential for antiviral prospection. The major component of Crotalus durissus terrificus venom is a heterodimeric complex called crotoxin, which is constituted by an inactive peptide (crotapotin) and a phospholipase A2 (PLA2-CB). We showed previously the antiviral effect of PLA2-CB against dengue virus, yellow fever virus and other enveloped viruses. The aims of this study were to express two PLA2-CB isoforms in a prokaryotic system and to evaluate their virucidal effects. The sequences encoding the PLA2-CB isoforms were optimized and cloned into a plasmid vector (pG21a) for recombinant protein expression. The recombinant proteins were expressed in the E. coli BL21(DE3) strain as insoluble inclusion bodies; therefore, the purification was performed under denaturing conditions, using urea for protein solubilization. The solubilized proteins were applied to a nickel affinity chromatography matrix for binding. The immobilized recombinant proteins were subjected to an innovative protein refolding step, which consisted of the application of a decreasing linear gradient of urea and dithiothreitol (DTT) concentrations in combination with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS) as a protein stabilizer. The refolded recombinant proteins showed phospholipase activity and virucidal effects against chikungunya virus, dengue virus, yellow fever virus and Zika virus.
TL;DR: A straightforward protocol for cholesterol oxidase production which can be performed in any laboratory is reported, and the optimum pH and optimum temperature for the enzyme activity were determined.
Abstract: Cholesterol oxidase is a bifunctional bacterial flavoenzyme which catalyzes oxidation and isomerization of cholesterol. This valuable enzyme has attracted a great deal of attention because of its wide application in the clinical laboratory, synthesis of steroid derived drugs, food industries, and its potentially insecticidal activity. Therefore, development of an efficient protocol for overproduction of cholesterol oxidase could be valuable and beneficial in this regard. The present study examined the role of various parameters (host strain, culture media, induction time, isopropyl s-D-1-thiogalactopyranoside concentration, as well as post-induction incubation time and temperature) on over-expression of cholesterol oxidase from Chromobacterium sp. DS1. Applying the optimized protocol, the yield of recombinant cholesterol oxidase significantly increased from 92 U/L to 2115 U/L. Under the optimized conditions, the enzyme was produced on a large-scale, and overexpressed cholesterol oxidase was purified from cell lysate by column nickel affinity chromatography. Km and Vmax values of the purified enzyme for cholesterol were estimated using Lineweaver-Burk plot. Further, the optimum pH and optimum temperature for the enzyme activity were determined. This study reports a straightforward protocol for cholesterol oxidase production which can be performed in any laboratory.
TL;DR: The use of peptide ligand HWRGWV and its cognate sequences to develop affinity adsorbents that compete with Protein A in terms of binding capacity and quality of the eluted product demonstrate the potential of peptides as alternative to Protein A for the purification of therapeutic antibodies.
Abstract: This work presents the use of peptide ligand HWRGWV and its cognate sequences to develop affinity adsorbents that compete with Protein A in terms of binding capacity and quality of the eluted product. First, the peptide ligand was conjugated to crosslinked agarose resins (WorkBeads) at different densities and using different spacer arms. The optimization of ligand density and display resulted in values of static and dynamic binding capacity of 85 mg/mL and 65 mg/mL, respectively. A selected peptide-WorkBeads adsorbent was utilized for purifying Mabs from Chinese Hamster Ovary (CHO) cell culture supernatants. The peptide-WorkBeads adsorbent was found able to withstand sanitization with strong alkaline solutions (0.5 M NaOH). The purity of the eluted product was consistently higher than 95%, with logarithmic removal value (LRV) of 1.5 for host cell proteins (HCPs) and 4.0 for DNA. HCP clearance was significantly improved by adding a post-load washing step with either 0.1 M Tris HCl pH 9 or 1 M NaCl. The cognate peptide of HWRGWV, constructed by replacing arginine (R) with citrulline, further increased the HCP LRV to 2.15. The peptide-based adsorbent also showed a remarkable performance in terms of removal of Mab aggregates; unlike Protein A, in fact, HWRGWV was found to bind only monomeric IgG. Collectively, these results demonstrate the potential of peptide-based adsorbents as alternative to Protein A for the purification of therapeutic antibodies.
TL;DR: The results showed that boric acid increased the G6PD enzyme activity while the mercury ions that inhibited the enzyme activity were noncompetitive.
Abstract: The aim of this study was to investigate the effects of mercury chloride and boric acid on rat (Wistar albino) erythrocyte: glucose 6-phosphate dehydrogenase (G6PD), 6-phosphoglucona-te dehydrogenase (6PGD), thioredoxin reductase (TrxR), glutathione reductase (GR) and glutathione S-transferase (GST) enzymes in vivo, and the rat erythrocyte G6PD enzyme in vitro. In the in vivo study, 24 male rats were divated into three different groups: control (C), mercury chloride (M), and mercury chloride + boric acid (M + BA). At the completion of this study, a significant degree of inhibition for both G6PD and GST enzyme activity was observed in the M groups when compared to the C group (p < 0.05), and no significant effect was observed in the 6PGD enzyme. However, there was significantly increased TrxR and GR enzyme activity of both the M and M + BA groups (p < 0.05). In the in vitro study, the G6PD enzyme from rat erythrocytes was purified with 2′,5′-ADP Sepharose-4B affinity chromatography, and the effect of both mercury chloride and boric acid on the enzyme activity was investigated. The results showed that boric acid increased the G6PD enzyme activity while the mercury ions that inhibited the enzyme activity (IC50 values of 346 μM and Ki values of 387 μM) were noncompetitive.
TL;DR: The aim of this study was to identify new gluten-degrading microbial enzymes with the potential to reduce gluten immunogenicity by neutralizing its antigenic epitopes, and the new enzyme was named PEP 2RA3.
Abstract: Gluten is a complex of proteins present in barley, wheat, rye and several varieties of oats that triggers celiac disease in genetically predisposed subjects. Gluten is notoriously difficult to digest by mammalian proteolytic enzymes and therefore, proline-rich digestion-resistant peptides contain multiple immunogenic epitopes. Prolyl endopeptidases (PEP) hydrolyse internal proline residues on the carboxyl side of peptides and have been proposed for food gluten detoxification and as oral enzyme supplementation for celiacs. The aim of this study was to identify new gluten-degrading microbial enzymes with the potential to reduce gluten immunogenicity by neutralizing its antigenic epitopes. Using a gluten-degrading colony screening approach, a bacterial isolate (2RA3) displaying the highest glutenase activity was selected, characterized and its genome completely sequenced. The identification through 16S rDNA gene sequencing showed a 99,1% similarity to Chryseobacterium taeanense. Hydrolysis of gluten immunogenic peptides (GIP) was further monitored, over a 48-hour period, by colony encapsulation in gliadin-containing microspheres, followed by detection with the G12 anti-GIP monoclonal antibody. Glutenase activity was detected in the extracellular medium of 2RA3 cultures, where gel electrophoresis and gliadin zymography revealed the presence of a ~50 kDa gluten-degrading enzyme. Nano-ESI-Q-TOF of the excised active band identified 7 peptides contained in the protein product predicted for an open reading frame (ORF) in the 2RA3 genome. Based on sequence similarity to the PEP family, the new enzyme was named PEP 2RA3. The PEP 2RA3 coding sequence was PCR-amplified from C. taeanense 2RA3, cloned and expressed in Escherichia coli as a C-terminally His-tagged recombinant protein and purified by Ni-NTA affinity chromatography. The recombinant protein, with predicted molecular mass and isoelectric point of 78.95 kDa and 6.8, respectively, shows PEP activity with standard chromogenic substrates, works optimally at pH 8.0 and 30°C and remains stable at pH 6.0 and 50°C, indicating a potential use in gluten-containing food process applications. The ability of the recombinant enzyme to degrade GIP in beer into smaller peptides was confirmed.
TL;DR: This report details the protocol for tandem affinity purification (TAP) primarily based on the use of the FLAG and HA peptide epitopes, with a particular emphasis on factors affecting yield and specificity.
Abstract: Affinity purification followed by mass spectrometry has become the technique of choice to identify binding partners in biochemical complexes isolated from a physiologic cellular context. In this report we detail our protocol for tandem affinity purification (TAP) primarily based on the use of the FLAG and HA peptide epitopes, with a particular emphasis on factors affecting yield and specificity, as well as steps to implement an automated version of the TAP procedure. © 2019 by John Wiley & Sons, Inc.
TL;DR: Two proteins that have not been previously reported in the secretomes of Trichoderma are described, a glycosyltransferase (six-harpin) and a galactose oxidase, belonging to the class of auxiliary activities (AA), classified as an AA subfamily AA5-2.
Abstract: Protein glycosylation is one of the most studied post-translational modifications and has received considerable attention for its critical role in the cell biology of eukaryotic cells. The genus Trichoderma has been extensively studied in the biocontrol of soil-borne fungal phytopathogens. The aim of this study was to identify the proteins secreted from Trichoderma harzianum after interacting with the cell walls of two phytopathogens, Sclerotinia sclerotiorum and Fusarium oxysporum. This study used N-glycoprotein enrichment with a concanavalin A (Con A) affinity column, staining detection differential SDS-PAGE, sequencing by mass spectrometric, and protein identification by comparison with the NCBI database to detect the protein expression of the two resulting secretome samples. The majority of the proteins found in both enriched secretomes belonged to a specific class of carbohydrate-active enzymes (CAZymes), within which glycosyl hydrolases (GHs), glycosyltransferases (GTs), and auxiliary activities (AAs) were identified. In this study was described two proteins that have not been previously reported in the secretomes of Trichoderma, a glycosyltransferase (six-harpin) and a galactose oxidase, belonging to the class of auxiliary activities (AA), classified as an AA subfamily AA5-2.The expression pattern of gene encoding to 17 identified proteins, evaluated by real-time quantitative PCR (RT-qPCR), showed significant difference of expression of some GHs and proteases, suggesting a specific gene expression regulation by T. harzianum in presence of different cell walls of two phytopathogens.
TL;DR: This thermostable β-xylanase revealed remarkable properties, which make it an encouraging candidate for various industrial applications especially in the alteration of renewable biomaterials into ethanol production, and biofuels from lignocellulosics has acknowledged much devotion subsequently in the last decade.
Abstract: The gene of a β-xylanase (Tnap_0700) was cloned from a hyperthermophilic Thermotoga naphthophila strain ATCC BAA-489 and expressed in Escherichia coli BL21 (DE3) via pET-21a (+) as an expression vector. The growth steps were upgraded for highest β-xylanase expression via several factors, for example, IPTG concentration, time of induction, pH, and temperature. The pH and temperature optima for the extreme expression of β-xylanase were 7.0 pH and 37 °C, correspondingly. Recombinant enzyme purified by heat treatment process, then later by immobilized metal ion affinity chromatography. Molecular mass of the purified β-xylanase was 38 kDa observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at room temperature for 30 days. It exhibited high stability over wide series of temperature 50-90 °C and pH 4.0-9.0 upon the addition of 1 mM Ca+2 and reduced in the existence of Cu+2 and EDTA. The addition of about 10-30% different organic solvents have no considerable effect on enzyme. However, SDSF and urea acting as an inhibitor leads to decrease in the enzyme activity. The β-xylanase enzyme was active to hydrolyze xylan from beechwood forming xylose. Thermostable β-xylanase causes the breakdown of complex carbohydrates into monosaccharide components. This thermostable β-xylanase revealed remarkable properties, which make it an encouraging candidate for various industrial applications especially in the alteration of renewable biomaterials into ethanol production, and biofuels from lignocellulosics has acknowledged much devotion subsequently in the last decade.
TL;DR: In this paper, Arabidopsis thaliana protein fractions were used to identify those differentially regulated following ergosterol treatment; additionally, they were subjected to affinity-based chromatography enrichment strategies to capture and categorize ergosterols-interacting candidate proteins using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).
Abstract: The impact of fungal diseases on crop production negatively reflects on sustainable food production and overall economic health. Ergosterol is the major sterol component in fungal membranes and regarded as a general elicitor or microbe-associated molecular pattern (MAMP) molecule. Although plant responses to ergosterol have been reported, the perception mechanism is still unknown. Here, Arabidopsis thaliana protein fractions were used to identify those differentially regulated following ergosterol treatment; additionally, they were subjected to affinity-based chromatography enrichment strategies to capture and categorize ergosterol-interacting candidate proteins using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Mature plants were treated with 250 nM ergosterol over a 24 h period, and plasma membrane-associated fractions were isolated. In addition, ergosterol was immobilized on two different affinity-based systems to capture interacting proteins/complexes. This resulted in the identification of defense-related proteins such as chitin elicitor receptor kinase (CERK), non-race specific disease resistance/harpin-induced (NDR1/HIN1)-like protein, Ras-related proteins, aquaporins, remorin protein, leucine-rich repeat (LRR)- receptor like kinases (RLKs), G-type lectin S-receptor-like serine/threonine-protein kinase (GsSRK), and glycosylphosphatidylinositol (GPI)-anchored protein. Furthermore, the results elucidated unknown signaling responses to this MAMP, including endocytosis, and other similarities to those previously reported for bacterial flagellin, lipopolysaccharides, and fungal chitin.
TL;DR: Ultra-performance liquid chromatography - tandem mass spectrometer (UPLC-MS) and monosaccharide composition analyses demonstrated that PpPel9a worked as an endo-pectate lyase, which acted primarily on the HG domain of citrus pectin.
Abstract: Pectate lyases play an important role in pectin degradation, and therefore are highly useful in the food and textile industries. Here, we report on the cloning of an alkaline pectate lyase gene (pppel9a) from Paenibacillus polymyxa KF-1. The full-length gene (1350 bp) encodes for a 449-residue protein that belongs to the polysaccharide lyase family 9 (PL9). Recombinant PpPel9a produced in Escherichia coli was purified to electrophoretic homogeneity in a single step using Ni2+-NTA affinity chromatography. The enzyme activity of PpPel9a (apparent molecular weight of 45.3 kDa) was found to be optimal at pH 10.0 and 40 °C, with substrate preference for homogalacturonan type (HG) pectins vis-a-vis rhamnogalacturonan-I (RG-I) type pectins. Using HG-type pectins as substrate, PpPel9a showed greater activity with de-esterified HGs. In addition, PpPel9a was active against water-soluble pectins isolated from different plants. Using this lyase, we degraded citrus pectin, purified fractions using Diethylaminoethyl (DEAE)-sepharose column chromatography, and characterized the main fraction MCP-0.3. High-performance gel permeation chromatography (HPGPC) analysis showed that the molecular mass of citrus pectin (~230.2 kDa) was reduced to ~24 kDa upon degradation. Ultra-performance liquid chromatography - tandem mass spectrometer (UPLC-MS) and monosaccharide composition analyses demonstrated that PpPel9a worked as an endo-pectate lyase, which acted primarily on the HG domain of citrus pectin. In vitro testing showed that the degradation product MCP-0.3 significantly promotes the growth of Lactobacillus plantarum and L. rhamnosus. In this regard, the enzyme has potential in the preparation of pharmacologically active pectin products.
TL;DR: Recombinant enzymes were used for bio-mineralization based conversion of atmospheric CO2 into valuable calcite in presence of enzymes and functional domain analysis of both CA proteins showed conserved region of respective proteins.
TL;DR: Applied applicability of FcɤRIIIa AC-MS is extended to Fab glycosylated IgG, i.e., cetuximab, by significantly reducing ambiguities in glycoform assignment vs. intact analysis.
Abstract: Fcɤ receptors (FcɤR) mediate key functions in immunological responses. For instance, FcɤRIIIa is involved in antibody-dependent cell-mediated cytotoxicity (ADCC). FcɤRIIIa interacts with the fragment crystallizable (Fc) of immunoglobulin G (IgG). This interaction is known to be highly dependent on IgG Fc glycosylation. Thus, the impact of glycosylation features on this interaction has been investigated in several studies by numerous analytical and biochemical techniques. FcɤRIIIa affinity chromatography (AC) hyphenated to mass spectrometry (MS) is a powerful tool to address co-occurring Fc glycosylation heterogeneity of monoclonal antibodies (mAbs). However, MS analysis of mAbs at the intact level may provide limited proteoform resolution, for example, when additional heterogeneity is present, such as antigen-binding fragment (Fab) glycosylation. Therefore, we investigated middle-up approaches to remove the Fab and performed AC-MS on the IgG Fc to evaluate its utility for FcɤRIIIa affinity assessment compared to intact IgG analysis. We found the protease Kgp to be particularly suitable for a middle-up FcɤRIIIa AC-MS workflow as demonstrated for the Fab glycosylated cetuximab. The complexity of the mass spectra of Kgp digested cetuximab was significantly reduced compared to the intact level while affinity was fully retained. This enabled a reliable assignment and relative quantitation of Fc glycoforms in FcɤRIIIa AC-MS. In conclusion, our workflow allows a functional separation of differentially glycosylated IgG Fc. Consequently, applicability of FcɤRIIIa AC-MS is extended to Fab glycosylated IgG, i.e., cetuximab, by significantly reducing ambiguities in glycoform assignment vs. intact analysis.
01 May 2019
TL;DR: The study concluded that TcSL showed anti-nociceptive and anti-inflammatory activities and thus poised to be a good bio-active peptide for the design of novel anti- inflammatory therapy.
Abstract: The anti-nociceptive and anti-inflammatory activity of the lectin purified from Tetracarpidium conophorum seeds were investigated. The lectin was purified from the phosphate buffered saline crude extract of the seeds using gel filtration and affinity chromatography techniques. Hemagglutination assay and hapten-sugar inhibition test were carried out to ascertain the blood group and sugar specificities of the lectin respectively. Anti-nociceptive and anti-inflammatory activities of the lectin were evaluated (in-vivo) with standard models including formalin licking test, carrageenan-induced paw edema test and peritonitis model. A yield, purification fold and subunit molecular weight of 27%, 17 and 34 kDa respectively were obtained for the purified lectin from T. conophorum seed (TcSL). The lactose/galactose specific TcSL showed significant inhibition (p The study concluded that TcSL showed anti-nociceptive and anti-inflammatory activities and thus poised to be a good bio-active peptide for the design of novel anti-inflammatory therapy.
TL;DR: A tag-free affinity method that employs functional selection interactions to a broad range of proteins and the competence of the PCNA-Agarose column to purify non-PCNA binding proteins is shown.
TL;DR: A method, based on the binding valency to Protein L resin, of separating proteins during the elution step by simply controlling the conductivity at low pH is reported, showing that the method efficiently separated targeted antibodies from mis-paired and aggregated species.
Abstract: The complex molecular formats of recent therapeutic antibodies, including bispecific antibodies, antibody fragments, and other fusion proteins, makes the task of purifying the desired molecules in a limited number of purification steps more and more challenging. Manufacturing these complicated biologics can be substantially improved in the affinity capture stage if the simple bind-and-elute mode is accompanied by targeted removal of the impurities, such as mis-paired antibodies and oligomers or aggregates. Here, we report a method, based on the binding valency to Protein L resin, of separating proteins during the elution step by simply controlling the conductivity at low pH. We show that the method efficiently separated targeted antibodies from mis-paired and aggregated species. Notably, the number of Protein L binding sites can be built into the molecule by design to facilitate the purification. This method may be useful for purifying various antibody formats at laboratory and manufacturing scales.