Showing papers on "Affinity chromatography published in 2022"
TL;DR: In this article, the RBD203-N1 yeast expression construct was used to produce a recombinant protein capable of eliciting a robust immune response and protection in mice against SARS-CoV-2 challenge infections.
13 citations
TL;DR: In this paper , the RBD203-N1 yeast expression construct was used to produce a recombinant protein capable of eliciting a robust immune response and protection in mice against SARS-CoV-2 challenge infections.
13 citations
TL;DR: In this paper , the expression and purification of the CsPP2-A1 gene in E. coli for further characteristic analysis was carried out. But the results showed that the CmPP2A1 in shake flask cultures was mainly expressed in the soluble form at 15 °C or in inclusion bodies at 37 °C.
10 citations
TL;DR: In this article , the potential of heparin affinity chromatography (HAC) to purify neural stem cell-derived EVs as part of a multistep process was evaluated and it was found that HAC could remove on average 98.8% and 99.0% of residual protein and DNA respectively.
9 citations
TL;DR: An ad hoc functionalized, universal support is conceived on which both the on‐support elongation and deprotection of the G4‐forming oligonucleotides can be performed, along with the successive affinity chromatography‐based assay, renamed as G‐quadruplex on Controlled Pore Glass (G4‐CPG) assay.
Abstract: Abstract DNA G‐quadruplexes (G4s) are key structures for the development of targeted anticancer therapies. In this context, ligands selectively interacting with G4s can represent valuable anticancer drugs. Aiming at speeding up the identification of G4‐targeting synthetic or natural compounds, we developed an affinity chromatography‐based assay, named G‐quadruplex on Oligo Affinity Support (G4‐OAS), by synthesizing G4‐forming sequences on commercially available polystyrene OAS. Then, due to unspecific binding of several hydrophobic ligands on nude OAS, we moved to Controlled Pore Glass (CPG). We thus conceived an ad hoc functionalized, universal support on which both the on‐support elongation and deprotection of the G4‐forming oligonucleotides can be performed, along with the successive affinity chromatography‐based assay, renamed as G‐quadruplex on Controlled Pore Glass (G4‐CPG) assay. Here we describe these assays and their applications to the screening of several libraries of chemically different putative G4 ligands. Finally, ongoing studies and outlook of our G4‐CPG assay are reported.
7 citations
TL;DR: In this article , a bispecific monoclonal antibody (BsMAb) was used to detect aflatoxin B1 (AFB1) and ochratoxin A (OTA) from food samples.
Abstract: A novel and efficient immunoaffinity column (IAC) based on bispecific monoclonal antibody (BsMAb) recognizing aflatoxin B1 (AFB1) and ochratoxin A (OTA) was prepared and applied in simultaneous extraction of AFB1 and OTA from food samples and detection of AFB1/OTA combined with ic-ELISA (indirect competitive ELISA). Two deficient cell lines, hypoxanthine guanine phosphoribosyl-transferase (HGPRT) deficient anti-AFB1 hybridoma cell line and thymidine kinase (TK) deficient anti-OTA hybridoma cell line, were fused to generate a hybrid-hybridoma producing BsMAb against AFB1 and OTA. The subtype of the BsMAb was IgG1 via mouse antibody isotyping kit test. The purity and molecular weight of BsMAb were confirmed by SDS-PAGE method. The cross-reaction rate with AFB2 was 37%, with AFG1 15%, with AFM1 48%, with AFM2 10%, and with OTB 36%. Negligible cross-reaction was observed with other tested compounds. The affinity constant (Ka) was determined by ELISA. The Ka (AFB1) and Ka (OTA) was 2.43 × 108 L/mol and 1.57 × 108 L/mol, respectively. Then the anti-AFB1/OTA BsMAb was coupled with CNBr-Sepharose, and an AFB1/OTA IAC was prepared. The coupling time and elution conditions of IAC were optimized. The coupling time was 1 h with 90% coupling rate, the eluent was methanol–water (60:40, v:v, pH 2.3) containing 1 mol/L NaCl, and the eluent volume was 4 mL. The column capacities of AFB1 and OTA were 165.0 ng and 171.3 ng, respectively. After seven times of repeated use, the preservation rates of column capacity for AFB1 and OTA were 69.3% and 68.0%, respectively. The ic-ELISA for AFB1 and OTA were applied combined with IAC. The IC50 (50% inhibiting concentration) of AFB1 was 0.027 ng/mL, the limit of detection (LOD) was 0.004 ng/mL (0.032 µg/kg), and the linear range was 0.006 ng/mL~0.119 ng/mL. The IC50 of OTA was 0.878 ng/mL, the LOD was 0.126 ng/mL (1.008 µg/kg), and the linear range was 0.259 ng/mL~6.178 ng/mL. Under optimum conditions, corn and wheat samples were pretreated with AFB1-OTA IAC. The recovery rates of AFB1 and OTA were 95.4%~105.0% with ic-ELISA, and the correlations between the detection results and LC-MS were above 0.9. The developed IAC combined with ic-ELISA is reliable and could be applied to the detection of AFB1 and OTA in grains.
7 citations
TL;DR: In this paper, a metal affinity-immobilized magnetic liposome (MA-IML) was used to adsorb ACE inhibitory peptides from lizard fish proteins hydrolysates.
7 citations
TL;DR: In this article , the authors examined the purification of Trastuzumab by cation-exchange z 2 laterally-fed membrane chromatography (or z 2 LFMC).
7 citations
TL;DR: Based on bioinformatic predictions, the S5 and S6 proteins in this study were produced, including S527-343, S 527-172, S5196-272, S630-317, S 630-190, and S 6200-317.
6 citations
TL;DR: In this article , a novel separation strategy for exosomes is proposed based on the specific interaction between immobilized peptide ligands and phosphatidylserine moieties which are highly abundant on the surface of exosome.
6 citations
TL;DR: In this paper , a novel in situ synthesis membrane protein affinity chromatography (iSMAC) method was developed utilizing cell-free protein expression (CFE) and covalent immobilized affinity Chromatography, which achieved efficient in- situ synthesis and unidirectional insertion of MPs into liposomes in the stationary phase.
01 Jan 2022
TL;DR: This chapter is describing methods to recombinantly express and purify galectins using three different methods of affinity purification, i.e., lactosyl-Sepharose chromatography for fungal galectin Coprinopsis cinereaGalectin 2 (CGL2), nickel-chromatography for histidine-tagged human galECTin-7, and glutathione-SepHarose chromation for Glutathione S-transferase-tagging (
TL;DR: An ensemble of peptide ligands that target the HCPs in Chinese hamster ovary (CHO) cell culture fluids and enable mAb purification via flow‐through affinity chromatography are introduced and their integration into LigaGuard™, an affinity adsorbent featuring an equilibrium binding capacity.
Abstract: The growth of advanced analytics in manufacturing monoclonal antibodies (mAbs) has highlighted the challenges associated with the clearance of host cell proteins (HCPs). Of special concern is the removal of “persistent” HCPs, including immunogenic and mAb‐degrading proteins, that co‐elute from the Protein A resin and can escape the polishing steps. Responding to this challenge, we introduced an ensemble of peptide ligands that target the HCPs in Chinese hamster ovary (CHO) cell culture fluids and enable mAb purification via flow‐through affinity chromatography. This study describes their integration into LigaGuard™, an affinity adsorbent featuring an equilibrium binding capacity of ~30 mg of HCPs per mL of resin as well as dynamic capacities up to 16 and 22 mg/ml at 1‐ and 2‐min residence times, respectively. When evaluated against cell culture harvests with different mAb and HCP titers and properties, LigaGuard™ afforded high HCP clearance, with logarithmic removal values (LRVs) up to 1.5, and mAb yield above 90%. Proteomic analysis of the effluents confirmed the removal of high‐risk HCPs, including cathepsins, histones, glutathione‐S transferase, and lipoprotein lipases. Finally, combining LigaGuard™ for HCP removal with affinity adsorbents for product capture afforded a global mAb yield of 85%, and HCP and DNA LRVs > 4.
TL;DR: In this paper, a two-stage method was proposed to enhance the expression and purification of recombinant Cusativin and uridine endoribonucleases using amylose affinity chromatography and 6xHIS tags.
TL;DR: In this paper , two different carbohydrate-binding modules (CBM3 and CBM9) have been successfully fused to an alcohol dehydrogenase from Saccharomyces cerevisiae, which has been produced in bench-scale reactor using an auxotrophic M15-derived E. coli strain, following a fed-batch strategy with antibiotic-free medium.
Abstract: The feasibility of biochemical transformation processes is usually greatly dependent on biocatalysts cost. Therefore, immobilizing and reusing biocatalysts is an approach to be considered to bring biotransformations closer to industrial feasibility, since it does not only allow to reuse enzymes but can also improve their stability towards several reaction conditions. Carbohydrate-Binding Modules (CBM) are well-described domains involved in substrate binding which have been already used as purification tags.In this work, two different Carbohydrate-Binding Modules (CBM3 and CBM9) have been successfully fused to an alcohol dehydrogenase from Saccharomyces cerevisiae, which has been produced in bench-scale reactor using an auxotrophic M15-derived E. coli strain, following a fed-batch strategy with antibiotic-free medium. Around 40 mg·g- 1 DCW of both fusion proteins were produced, with a specific activity of > 65 AU·mg- 1. Overexpressed proteins were bound to a low-cost and highly selective cellulosic support by one-step immobilization/purification process at > 98% yield, retaining about a 90% of initial activity. Finally, the same support was also used for protein purification, aiming to establish an alternative to metal affinity chromatography, by which CBM9 tag proved to be useful, with a recovery yield of > 97% and 5-fold increased purity grade.CBM domains were proved to be suitable for one-step immobilization/purification process, retaining almost total activity offered. However, purification process was only successful with CBM9.
TL;DR: In this article , a novel codon optimized β-glucosidase gene from Trichoderma reesei QM6a was cloned and expressed in three strains of Escherichia coli (E. coli).
TL;DR: In this article , two different carbohydrate-binding modules (CBM3 and CBM9) have been successfully fused to an alcohol dehydrogenase from Saccharomyces cerevisiae, which has been produced in bench-scale reactor using an auxotrophic M15-derived E. coli strain, following a fed-batch strategy with antibiotic-free medium.
Abstract: The feasibility of biochemical transformation processes is usually greatly dependent on biocatalysts cost. Therefore, immobilizing and reusing biocatalysts is an approach to be considered to bring biotransformations closer to industrial feasibility, since it does not only allow to reuse enzymes but can also improve their stability towards several reaction conditions. Carbohydrate-Binding Modules (CBM) are well-described domains involved in substrate binding which have been already used as purification tags.In this work, two different Carbohydrate-Binding Modules (CBM3 and CBM9) have been successfully fused to an alcohol dehydrogenase from Saccharomyces cerevisiae, which has been produced in bench-scale reactor using an auxotrophic M15-derived E. coli strain, following a fed-batch strategy with antibiotic-free medium. Around 40 mg·g- 1 DCW of both fusion proteins were produced, with a specific activity of > 65 AU·mg- 1. Overexpressed proteins were bound to a low-cost and highly selective cellulosic support by one-step immobilization/purification process at > 98% yield, retaining about a 90% of initial activity. Finally, the same support was also used for protein purification, aiming to establish an alternative to metal affinity chromatography, by which CBM9 tag proved to be useful, with a recovery yield of > 97% and 5-fold increased purity grade.CBM domains were proved to be suitable for one-step immobilization/purification process, retaining almost total activity offered. However, purification process was only successful with CBM9.
TL;DR: In this article , a two-stage method was proposed to enhance the expression and purification of recombinant Cusativin and uridine endoribonucleases using amylose affinity chromatography and 6xHIS tags.
TL;DR: In this article, a novel codon optimized β-glucosidase gene from Trichoderma reesei QM6a was cloned and expressed in three strains of Escherichia coli (E. coli).
TL;DR: A multi-step approach for the expression and purification of homodimeric, fully active, histidine-tagged recombinant gremlin-1, using mammalian HEK293T cells enables obtaining a highly pure recombinant dimeric g Kremlin-1 protein, exhibiting both BMP antagonist and potent VEGFR2 agonist activities.
Abstract: Gremlin-1 is a secreted cystine-knot protein that acts as an antagonist of bone morphogenetic proteins (BMPs), and as a ligand of heparin and the vascular endothelial growth factor receptor 2 (VEGFR2), thus regulating several physiological and pathological processes, including embryonic development, tissue fibrosis and cancer. Gremlin-1 exerts all these biological activities only in its homodimeric form. Here, we propose a multi-step approach for the expression and purification of homodimeric, fully active, histidine-tagged recombinant gremlin-1, using mammalian HEK293T cells. Ion metal affinity chromatography (IMAC) of crude supernatant followed by heparin-affinity chromatography enables obtaining a highly pure recombinant dimeric gremlin-1 protein, exhibiting both BMP antagonist and potent VEGFR2 agonist activities.
TL;DR: A simple, fast, and specific capability for the prediction of high‐mannose content in samples compared with traditional glycan profiling by 2‐aminobenzamide or mass spectrometry‐based methods is offered.
Abstract: We developed a simple high‐performance liquid chromatography assay to monitor high‐mannose glycans in monoclonal antibodies by monitoring terminal alpha‐mannose as a surrogate marker. Analysis of glycan data of therapeutic monoclonal antibodies by 2‐aminobenzamide assay showed a linear relationship between high mannose and terminal mannose of Fc glycans. Concanavalin A has a strong affinity to alpha‐mannose in glycans of typical therapeutic monoclonal antibodies. To show that terminal mannose binds specifically to Concanavalin A column, exoglycosidase‐treated monoclonal antibodies were serially blended with untreated monoclonal antibodies. Linear responses of terminal‐mannose binding to the column and comparable data trending with high mannose levels by 2‐aminobenzamide assay confirmed that terminal‐mannose levels measured by the Concanavalin A column can be used as a surrogate for the prediction of high‐mannose levels in monoclonal antibodies. The assay offers a simple, fast, and specific capability for the prediction of high‐mannose content in samples compared with traditional glycan profiling by 2‐aminobenzamide or mass spectrometry‐based methods. When the Concanavalin A column was coupled with protein A column for purification of antibodies from cell culture samples in a fully automated two‐dimensional analysis, high‐mannose data could be relayed to the manufacturing team in less than 30 min, allowing near‐real‐time monitoring of high‐mannose levels in the cell culture process.
TL;DR: In this paper, an easy pipeline for expression of SARS-CoV-2 nucleocapsid (N) protein in insect cells followed by its purification via affinity chromatography was described.
TL;DR: The ZZ-CSQ platform is rapid, recyclable, scalable, and cost-effective, and it shows antibody-purification performance superior or comparable to that of the standard affinity chromatography method.
Abstract: Affinity chromatography utilizing specific interactions between therapeutic proteins and bead-immobilized capturing agents is a standard method for protein purification, but its scalability is limited by long purification times, activity loss by the capturing molecules and/or purified protein, and high costs. Here, we report a platform for purifying therapeutic antibodies via affinity precipitation using the endogenous calcium ion-binding protein, calsequestrin (CSQ), which undergoes a calcium ion-dependent phase transition. In this method, ZZ-CSQ fusion proteins with CSQ and an affinity protein (Z domain of protein A) capture antibodies and undergo multimerization and subsequent aggregation in response to calcium ions, enabling the antibody to be collected by affinity precipitation. After robustly validating and optimizing the performance of the platform, the ZZ-CSQ platform can rapidly purify therapeutic antibodies from industrial harvest feedstock with high purity (>97%) and recovery yield (95% ± 3%). In addition, the ZZ-CSQ platform outperforms protein A-based affinity chromatography (PAC) in removing impurities, yielding ∼20-fold less DNA and ∼4.8-fold less host cell protein (HCP) contamination. Taken together, this platform is rapid, recyclable, scalable, and cost-effective, and it shows antibody-purification performance superior or comparable to that of the standard affinity chromatography method.
TL;DR: In this paper , an easy pipeline for expression of SARS-CoV-2 nucleocapsid (N) protein in insect cells followed by its purification via affinity chromatography was described.
TL;DR: This review highlighted the recent trends in designing biomimetic affinity ligands and summarized their binding interactions with the target molecules with computational approaches.
Abstract: Affinity chromatography is a well-known method dependent on molecular recognition and is used to purify biomolecules by mimicking the specific interactions between the biomolecules and their substrates. Enzyme substrates, cofactors, antigens, and inhibitors are generally utilized as bioligands in affinity chromatography. However, their cost, instability, and leakage problems are the main drawbacks of these bioligands. Biomimetic affinity ligands can recognize their target molecules with high selectivity. Their cost-effectiveness and chemical and biological stabilities make these antibody analogs favorable candidates for affinity chromatography applications. Biomimetics applies to nature and aims to develop nanodevices, processes, and nanomaterials. Today, biomimetics provides a design approach to the biomimetic affinity ligands with the aid of computational methods, rational design, and other approaches to meet the requirements of the bioligands and improve the downstream process. This review highlighted the recent trends in designing biomimetic affinity ligands and summarized their binding interactions with the target molecules with computational approaches.
TL;DR: In this paper , N-terminal amino acid sequencing and liquid chromatography with quadrupole time-of-flight tandem mass spectrometry (LC/Q-TOF-MS) analysis revealed that the 90-kDa protein was lipovitellin heavy-chain (LvH), which is one of the proteolytically cleaved products of maternal vitellogenin and represents the main precursor of the egg yolk in teleosts, and the 30-kda protein was an Nterminal bit of LvH, suggesting that the latter protein plays an important role during embryogenesis in the flounder.
TL;DR: In this article , single-stranded DNA aptamers with binding affinity to Ole e 1, the major allergen of olive pollen, were selected using systematic evolution of ligands by exponential enrichment (SELEX) method.
TL;DR: In this paper , the first known non-affinity purification method for two S constructs, S_dF_2P and HexaPro, expressed in the mammalian cell line, CHO-DG44, was presented.
Abstract: The spike (S) glycoprotein of the pandemic virus, SARS-CoV-2, is a critically important target of vaccine design and therapeutic development. A high-yield, scalable, cGMP-compliant downstream process for the stabilized, soluble, native-like S protein ectodomain is necessary to meet the extensive material requirements for ongoing research and development. As of June 2021, S proteins have exclusively been purified using difficult-to-scale, low-yield methodologies such as affinity and size-exclusion chromatography. Herein we present the first known non-affinity purification method for two S constructs, S_dF_2P and HexaPro, expressed in the mammalian cell line, CHO-DG44. A high-throughput resin screen on the Tecan Freedom EVO200 automated bioprocess workstation led to identification of ion exchange resins as viable purification steps. The chromatographic unit operations along with industry-standard methodologies for viral clearances, low pH treatment and 20 nm filtration, were assessed for feasibility. The developed process was applied to purify HexaPro from a CHO-DG44 stable pool harvest and yielded the highest yet reported amount of pure S protein. Our results demonstrate that commercially available chromatography resins are suitable for cGMP manufacturing of SARS-CoV-2 Spike protein constructs. We anticipate our results will provide a blueprint for worldwide biopharmaceutical production laboratories, as well as a starting point for process intensification.
TL;DR: In this paper, a silkworm-derived norovirus-like particle (NoV-LPs) was extracted from fat body lysate by density gradient centrifugation and further purified using immobilized metal affinity chromatography.
TL;DR: A functional anti-human Tim-3 nanobody with high affinity in vitro is presented, successfully prepared and immunized a 4-year-old female alpaca with TIM-3 antigen.
Abstract: Monoclonal antibodies and antibody-derived biologics are essential tools for cancer research and therapy. The development of monoclonal antibody treatments for successful tumor-targeted therapies took several decades. A nanobody constructed by molecular engineering of heavy-chain-only antibody, which is unique in camel or alpaca, is a burgeoning tools of diagnostic and therapeutic in clinic. In this study, we immunized a 4-year-old female alpaca with TIM-3 antigen. Then, a VHH phage was synthesized from the transcriptome of its B cells by nested PCR as an intermediate library; the library selection for Tim-3 antigen is carried out in three rounds of translation. The most reactive colonies were selected by periplasmic extract monoclonal ELISA. The nanobody was immobilized by metal affinity chromatography (IMAC) purification with the use of a Ni-NTA column, SDS-PAGE, and Western blotting. Finally, the affinity of TIM3-specific nanobody was determined by ELISA. As results, specific 15 kD bands representing nanomaterials were observed on the gel and confirmed by Western blotting. The nanobody showed obvious specific immune response to Tim-3 and had high binding affinity. We have successfully prepared a functional anti-human Tim-3 nanobody with high affinity in vitro.