Showing papers on "Affinity chromatography published in 2021"

Production of trimeric SARS-CoV-2 spike protein by CHO cells for serological COVID-19 testing.

Abstract: We describe scalable and cost‐efficient production of full length, His‐tagged severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike glycoprotein trimer by Chinese hamster ovary (CHO) cells that can be used to detect SARS‐CoV‐2 antibodies in patient sera at high specificity and sensitivity. Transient production of spike in both human embryonic kidney (HEK) and CHO cells mediated by polyethyleneimine was increased significantly (up to 10.9‐fold) by a reduction in culture temperature to 32°C to permit extended duration cultures. Based on these data GS‐CHO pools stably producing spike trimer under the control of a strong synthetic promoter were cultured in hypothermic conditions with combinations of bioactive small molecules to increase yield of purified spike product 4.9‐fold to 53 mg/L. Purification of recombinant spike by Ni‐chelate affinity chromatography initially yielded a variety of co‐eluting protein impurities identified as host cell derived by mass spectrometry, which were separated from spike trimer using a modified imidazole gradient elution. Purified CHO spike trimer antigen was used in enzyme‐linked immunosorbent assay format to detect immunoglobulin G antibodies against SARS‐CoV‐2 in sera from patient cohorts previously tested for viral infection by polymerase chain reaction, including those who had displayed coronavirus disease 2019 (COVID‐19) symptoms. The antibody assay, validated to ISO 15189 Medical Laboratories standards, exhibited a specificity of 100% and sensitivity of 92.3%. Our data show that CHO cells are a suitable host for the production of larger quantities of recombinant SARS‐CoV‐2 trimer which can be used as antigen for mass serological testing.

Isolation, identification, and characterization of the human airway ligand for the eosinophil and mast cell immunoinhibitory receptor Siglec-8.

Abstract: Background The immunoinhibitory receptor Siglec-8 on the surface of human eosinophils and mast cells binds to sialic acid–containing ligands in the local milieu, resulting in eosinophil apoptosis, inhibition of mast cell degranulation, and suppression of inflammation. Siglec-8 ligands were found on postmortem human trachea and bronchi and on upper airways in 2 compartments, cartilage and submucosal glands, but they were surprisingly absent from the epithelium. We hypothesized that Siglec-8 ligands in submucosal glands and ducts are normally transported to the airway mucus layer, which is lost during tissue preparation. Objective Our aim was to identify the major Siglec-8 sialoglycan ligand on the mucus layer of human airways. Methods Human upper airway mucus layer proteins were recovered during presurgical nasal lavage of patients at a sinus clinic. Proteins were resolved by gel electrophoresis and blotted, and Siglec-8 ligands detected. Ligands were purified by size exclusion and affinity chromatography, identified by proteomic mass spectrometry, and validated by electrophoretic and histochemical colocalization. The affinity of Siglec-8 binding to purified human airway ligand was determined by inhibition of glycan binding. Results A Siglec-8-ligand with a molecular weight of approximately 1000 kDa was found in all patient nasal lavage samples. Purification and identification revealed deleted in malignant brain tumors 1 (DMBT1) (also known by the aliases GP340 and SALSA), a large glycoprotein with multiple O-glycosylation repeats. Immunoblotting, immunohistochemistry, and enzyme treatments confirmed that Siglec-8 ligand on the human airway mucus layer is an isoform of DMBT1 carrying O-linked sialylated keratan sulfate chains (DMBT1S8). Quantitative inhibition revealed that DMBT1S8 has picomolar affinity for Siglec-8. Conclusion A distinct DMBT1 isoform, DMBT1S8, is the major high-avidity ligand for Siglec-8 on human airways.

The SARS-CoV-2 receptor-binding domain expressed in Pichia pastoris as a candidate vaccine antigen

Abstract: 1.The effort to develop vaccines based on economically accessible technological platforms available by developing countries vaccine manufacturers is essential to extend the immunization to the whole world population and to achieve the desired herd immunity, necessary to end the COVID-19 pandemic. Here we report on the development of a SARS-CoV-2 receptor-binding domain (RBD) protein, expressed in yeast Pichia pastoris. The RBD was modified with addition of flexible N- and C-terminal amino acid extensions aimed to modulate the protein/protein interactions and facilitate protein purification. Fermentation with yeast extract culture medium yielded 30-40 mg/L. After purification by immobilized metal ion affinity chromatography and hydrophobic interaction chromatography, the RBD protein was characterized by mass-spectrometry, circular dichroism, and binding affinity to angiotensin-converting enzyme 2 (ACE2) receptor. The recombinant protein shows high antigenicity with convalescent human sera and also with sera from individuals vaccinated with the Pfizer-BioNTech mRNA or Sputnik V adenoviral-based vaccines. The RBD protein stimulates IFN{gamma}, IL-2, IL-6, IL-4, and TNF in mice secreting splenocytes from PBMC and lung CD3+ enriched cells. Immunogenicity studies with 50 {micro}g of the recombinant RBD formulated with alum, induce high levels of binding antibodies in mice and non-human primates, assessed by ELISA plates covered with RBD protein expressed in HEK293T cells. The mouse sera inhibited the RBD binding to ACE2 receptor in an in-vitro test and show neutralization of SARS-CoV-2 infection of Vero E6 cells. These data suggest that the RBD recombinant protein expressed in yeast P. pastoris is suitable as a vaccine candidate against COVID-19. HighlightsO_LIThe RBD protein (C-RBD-H6 PP) is expressed with high purity in P. pastoris. C_LIO_LIPhysico-chemical characterization confirms the right folding of the protein. C_LIO_LIThe recombinant protein shows high antigenicity with sera from convalescents. C_LIO_LIThe sera from animals inhibit the RBD-ACE2 binding and neutralize the virus. C_LIO_LIThe C-RBD-H6 protein stimulates IFN{gamma}, IL-2, IL-6, IL-4, and TNF in mice. C_LI

Serological Test to Determine Exposure to SARS-CoV-2: ELISA Based on the Receptor-Binding Domain of the Spike Protein (S-RBD N318-V510 ) Expressed in Escherichia coli .

Abstract: Massive worldwide serological testing for SARS-CoV-2 is needed to determine the extent of virus exposure in a particular region, the ratio of symptomatic to asymptomatic infected persons, and the duration and extent of immunity after infection To achieve this, the development and production of reliable and cost-effective SARS-CoV-2 antigens is critical We report the bacterial production of the peptide S-RBDN318-V510, which contains the receptor-binding domain of the SARS-CoV-2 spike protein (region of 193 amino acid residues from asparagine-318 to valine-510) of the SARS-CoV-2 spike protein We purified this peptide using a straightforward approach involving bacterial lysis, his-tag-mediated affinity chromatography, and imidazole-assisted refolding The antigen performances of S-RBDN318-V510 and a commercial full-length spike protein were compared in ELISAs In direct ELISAs, where the antigen was directly bound to the ELISA surface, both antigens discriminated sera from non-exposed and exposed individuals However, the discriminating resolution was better in ELISAs that used the full-spike antigen than the S-RBDN318-V510 Attachment of the antigens to the ELISA surface using a layer of anti-histidine antibodies gave equivalent resolution for both S-RBDN318-V510 and the full-length spike protein Results demonstrate that ELISA-functional SARS-CoV-2 antigens can be produced in bacterial cultures, and that S-RBDN318-V510 may represent a cost-effective alternative to the use of structurally more complex antigens in serological COVID-19 testing

Molecular docking of metal ion immobilized ligands to proteins in affinity chromatography.

Abstract: Immobilized metal ion affinity chromatography (IMAC) has become a widespread analytical and preparative separation method for therapeutic proteins, peptides nucleic acids, hormones, and enzymes. N-Methacryloyl-l-histidine Methyl Ester (MAH) monomer is recently used as a synthesized affinity ligand in IMAC. It is capable of chelating with many transition metal ions such as Zn2+ , Ni2+ , and Cu2+ ions through its histidine residue. In this way, proteins can bind selectively to these immobilized metal ions through MAH as a ligand in affinity chromatography. In this study, we applied the computational docking method on the interactions that occur between the MAH monomer and its complexes with Zn2+ ions as ligands and protein molecules as targets. MAH monomer was drawn and created using the Avogadro software as an optimization tool. Human insulin (Ins) molecule and horse heart cytochrome C (Cyt C) were selected as target proteins to interact with MAH monomer as affinity ligand. Automated docking software AutoDock v4.2 was used for docking of MAH monomer to Ins and Cyt C, respectively. The affinity ligand complexes with Zn2+ ions bound to one, two, and three moles of MAH were studied and compared separately. The lowest binding energies of Ins and Cyt C proteins in 1:1 mol ratio of MAH-Zn2+ were found as (-4.14) and (-4.92) kcal/mol, respectively.

Binding characteristics of staphylococcal protein A and streptococcal protein G for fragment crystallizable portion of human immunoglobulin G.

Abstract: In the wide array of physiological processes, protein-protein interactions and their binding are the most basal activities for achieving adequate biological metabolism. Among the studies on binding proteins, the examination of interactions between immunoglobulin G (IgG) and natural immunoglobulin-binding ligands, such as staphylococcal protein A (spA) and streptococcal protein G (spG), is essential in the development of pharmaceutical science, biotechnology, and affinity chromatography. The widespread utilization of IgG-spA/spG binding characteristics has allowed researchers to investigate these molecular interactions. However, the detailed binding strength of each ligand and the corresponding binding mechanisms have yet to be fully investigated. In this study, the authors analyzed the binding strengths of IgG-spA and IgG-spG complexes and identified the mechanisms enabling these bindings using molecular dynamics simulation, steered molecular dynamics, and advanced Poisson-Boltzmann Solver simulations. Based on the presented data, the binding strength of the spA ligand was found to significantly exceed that of the spG ligand. To find out which non-covalent interactions or amino acid sites have a dominant role in the tight binding of these ligands, further detailed analyses of electrostatic interactions, hydrophobic bonding, and binding free energies have been performed. In investigating their binding affinity, a relatively independent and different unbinding mechanism was found in each ligand. These distinctly different mechanisms were observed to be highly correlated to the protein secondary and tertiary structures of spA and spG ligands, as explicated from the perspective of hydrogen bonding.

Generation of cell-permeant recombinant human transcription factor GATA4 from E. coli

Abstract: Transcription factor GATA4 is expressed during early embryogenesis and is vital for proper development In addition, it is a crucial reprogramming factor for deriving functional cardiomyocytes and was recently identified as a tumor suppressor protein in various cancers To generate a safe and effective molecular tool that can potentially be used in a cell reprogramming process and as an anti-cancer agent, we have identified optimal expression parameters to obtain soluble expression of human GATA4 in E coli and purified the same to homogeneity under native conditions using immobilized metal ion affinity chromatography The identity of GATA4 protein was confirmed using western blotting and mass spectrometry Using circular dichroism spectroscopy, it was demonstrated that the purified recombinant protein has maintained its secondary structure, primarily comprising of random coils and α-helices Subsequently, this purified recombinant protein was applied to human cells and was found that it was non-toxic and able to enter the cells as well as translocate to the nucleus Prospectively, this cell- and nuclear-permeant molecular tool is suitable for cell reprogramming experiments and can be a safe and effective therapeutic agent for cancer therapy

One-step selective affinity purification and immobilization of His-tagged enzyme by recyclable magnetic nanoparticles.

Abstract: The NiFe2O4 magnetic nanoparticles (NF-MNPs) were prepared for one-step selective affinity purification and immobilization of His-tagged recombinant glucose dehydrogenase (GluDH). The prepared nanoparticles were characterized by a Fourier-transform infrared spectrophotometer and microscopy. The immobilization and purification of His-tagged GluDH on NF-MNPs were investigated. The optimal immobilization conditions were obtained that mixed cell lysis and carriers in a ratio of 0.13 in pH 8.0 Tris-HCl buffer at 30℃ and incubated for 2 h. The highest activity recovery and protein bindings were 71.39% and 38.50 μg mg-1 support, respectively. The immobilized GluDH exhibited high thermostability, pH-stability and it can retain more than 65% of the initial enzyme after 10 cycles for the conversion of glucose to gluconolactone. Comparing with a commercial Ni-NTA resin, the NF-MNPs displayed a higher specific affinity with His-tagged recombinant GluDH.

A New Approach for Affinity-Based Purification of Horseradish Peroxidase.

Abstract: We have developed efficient procedure for isolation of horseradish peroxidase (HRP) using aminobenzohydrazide-based affinity chromatography. Sepharose 4B-bounded aminobenzohydrazides are suitable for long-term use and large-scale purification. In this study, 26 aminobenzohydrazide derivatives were synthesized, characterized and defined as new HRP inhibitors. In addition, detailed inhibition effects of these molecules on HRP enzyme were investigated. Affinity matrix was formed by bonding aminobenzohydrazides, which exhibited inhibitory activity to sepharose-4B-l-tyrosine. HRP was isolated from crude homogenate in single step and purification factors were recorded as 1,151-fold (recovery of 8.5%) with 4-amino 3-bromo benzohydrazide and as 166.16-fold (recovery of 16.67 %) with 3-amino 4-chloro benzohydrazide.

Affinity Purification of Angiotensin Converting Enzyme Inhibitory Peptides from Wakame (Undaria Pinnatifida) Using Immobilized ACE on Magnetic Metal Organic Frameworks.

Abstract: Angiotensin-I-converting enzyme (ACE) inhibitory peptides derived from marine organism have shown a blood pressure lowering effect with no side effects. A new affinity medium of Fe3O4@ZIF-90 immobilized ACE (Fe3O4@ZIF-90-ACE) was prepared and used in the purification of ACE inhibitory peptides from Wakame (Undaria pinnatifida) protein hydrolysate (<5 kDa). The Fe3O4@ZIF-90 nanoparticles were prepared by a one-pot synthesis and crude ACE extract from pig lung was immobilized onto it, which exhibited excellent stability and reusability. A novel ACE inhibitory peptide, KNFL (inhibitory concentration 50, IC50 = 225.87 μM) was identified by affinity purification using Fe3O4@ZIF-90-ACE combined with reverse phase-high performance liquid chromatography (RP-HPLC) and MALDI-TOF mass spectrometry. Lineweaver–Burk analysis confirmed the non-competitive inhibition pattern of KNFL, and molecular docking showed that it bound at a non-active site of ACE via hydrogen bonds. This demonstrates that affinity purification using Fe3O4@ZIF-90-ACE is a highly efficient method for separating ACE inhibitory peptides from complex protein mixtures and the purified peptide KNFL could be developed as a functional food ingredients against hypertension.

Selecting Subpopulations of High-Quality Protein Conformers among Conformational Mixtures of Recombinant Bovine MMP-9 Solubilized from Inclusion Bodies.

Abstract: A detailed workflow to analyze the physicochemical characteristics of mammalian matrix metalloproteinase (MMP-9) protein species obtained from protein aggregates (inclusion bodies—IBs) was followed. MMP-9 was recombinantly produced in the prokaryotic microbial cell factories Clearcoli (an engineered form of Escherichia coli) and Lactococcus lactis, mainly forming part of IBs and partially recovered under non-denaturing conditions. After the purification by affinity chromatography of solubilized MMP-9, four protein peaks were obtained. However, so far, the different conformational protein species forming part of IBs have not been isolated and characterized. Therefore, with the aim to link the physicochemical characteristics of the isolated peaks with their biological activity, we set up a methodological approach that included dynamic light scattering (DLS), circular dichroism (CD), and spectrofluorometric analysis confirming the separation of subpopulations of conformers with specific characteristics. In protein purification procedures, the detailed analysis of the individual physicochemical properties and the biological activity of protein peaks separated by chromatographic techniques is a reliable source of information to select the best-fitted protein populations.

Advanced purification platform using circularly permuted caspase‐2 for affinity fusion‐tag removal to produce native fibroblast growth factor 2

Abstract: BACKGROUND: Recombinant proteins produced for use as biopharmaceuticals need to harbor their native N‐terminus. A drawback in expression of recombinant proteins as fusion proteins with an affinity fusion‐tag is that enzymatic or chemical processing is required to trim the artificial tag and release the true protein of interest. In many cases, however, this processing step generates an incorrect N‐terminus. RESULTS: Human fibroblast growth factor 2 (FGF2) was expressed as a fusion protein in Escherichia coli fed‐batch cultivations. The protein of interest (POI) carried an N‐terminal affinity fusion‐tag which enabled purification via affinity chromatography. After enzymatic removal of the affinity fusion‐tag with a circularly permuted human caspase‐2 (cpCasp2), the POI was further purified using subtractive affinity chromatography. Mass spectrometric analysis confirmed the authentic N‐terminus of the POI. The generated POI was highly pure with 42 ppm host cell protein, 3.7 μg mL⁻¹ dsDNA and ~ 1000 EU mL⁻¹ endotoxin. Only a small number of E. coli host cell proteins were co‐purified with the POI. Because of the high specificity of the novel protease cpCasp2, no off‐target cleavage could be observed. CONCLUSION: Our findings demonstrate that cpCasp2 can be used for the production of native proteins using a fusion‐protein process. This represents a first case study at large laboratory scale for the production of an industrially relevant protein. This technology constitutes the basis of a highly scalable cpCasp2‐based platform fusion protein process (CASPON technology) purification platform. © 2021 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

High complexity of Glutamine synthetase regulation in Methanosarcina mazei: Small protein 26 interacts and enhances glutamine synthetase activity.

Abstract: Small ORF (sORF)-encoded small proteins have been overlooked for a long time due to challenges in prediction and distinguishing between coding- and noncoding-predicted sORFs and in their biochemical detection and characterization. We report on the first biochemical and functional characterization of a small protein (sP26) in the archaeal model organism Methanosarcina mazei, comprising 23 amino acids. The corresponding encoding leaderless mRNA (spRNA26) is highly conserved on nucleotide level as well as on the coded amino acids within numerous Methanosarcina strains strongly arguing for a cellular function of the small protein. spRNA26 level is significantly enhanced under nitrogen limitation, but also under oxygen and salt stress conditions. Using heterologously expressed and purified sP26 in independent biochemical approaches [pull-down by affinity chromatography followed by MS analysis, reverse pull-down, microscale thermophoresis, size-exclusion chromatography, and nuclear magnetic resonance spectroscopy (NMR) analysis], we observed that sP26 interacts and forms complexes with M. mazei glutamine synthetase (GlnA1 ) with high affinity (app. KD = 0.76 µm± 0.29 µm). Moreover, seven amino acids were identified by NMR analysis to directly interact with GlnA1 . Upon interaction with sP26, GlnA1 activity is significantly stimulated, independently and in addition to the known activation by the metabolite 2-oxoglutarate (2-OG). Besides, strong interaction of sP26 with the PII-like protein GlnK1 was demonstrated (app. KD = 2.9 µm ± 0.9 µm). On the basis of these findings, we propose that in addition to 2-OG, sP26 enhances GlnA1 activity under nitrogen limitation most likely by stabilizing the dodecameric structure of GlnA1 .

Antibiotics as Inhibitor of Glutathione S-transferase: Biological Evaluation and Molecular Structure Studies.

Abstract: Background The glutathione S-transferases (GSTs) are family of enzymes that are notable for their role in phase II detoxification reactions. Antibiotics have been reported to have several adverse effects on the activity of the enzymes in mammals. Aim The aim of this study was structural and biochemical characterization of rat erythrocyte GST and understanding the effects of gentamicin, clindamycin, cefazolin, ampicillin and scopolamine butylbromide on the activity of human erythrocyte GST using rat as a model. Methods The enzyme was purified by GSH-agarose affinity chromatography. In vitro GST enzyme activity was measured at 25°C using CDNB as a model substrate. IC50 of drugs were measured by activity %-vs compound concentration graphs. Lineweaver-Burk graphs were drawn to determine the inhibition type and Ki constants for the drugs. The structure of the enzyme was predicted via Protein Homology/analogY Recognition Engine. Results In this study, GST was purified from rat erythrocyte with a specific activity of 6.3 EU/mg protein, 44 % yield and 115 fold. Gentamicin and clindamycin inhibited the enzymatic activity with IC50 of 1.69 and 6.9 mM and Ki of 1.70 and 2.36 mM, respectively. Ampicillin and scopolamine butylbromide were activator of the enzyme while the activity of the enzyme was insensitive to cefazolin. The enzyme was further characterized by homology modeling and sequence alignment revealing similarities with human GST. Conclusion Collectively, it could be concluded that gentamicin and clindamycin are the inhibitors of erythrocyte GST.

Recent Advances in Lectin-Based Affinity Sorbents for Protein Glycosylation Studies.

Abstract: Glycosylation is one of the most significant post-translational modifications occurring to proteins, since it affects some of their basic properties, such as their half-life or biological activity. The developments in analytical methodologies has greatly contributed to a more comprehensive understanding of the quantitative and qualitative characteristics of the glycosylation state of proteins. Despite those advances, the difficulty of a full characterization of glycosylation still remains, mainly due to the complexity of the glycoprotein and/or glycopeptide mixture especially when they are present in complex biological samples. For this reason, various techniques that allow a prior selective enrichment of exclusively glycosylated proteins or glycopeptides have been developed in the past and are coupled either on- or off- line with separation and detection methods. One of the most commonly implemented enrichment methods includes the use of lectin proteins immobilized on various solid supports. Lectins are a group of different, naturally occurring proteins that share a common characteristic, which concerns their affinity for specific sugar moieties of glycoproteins. This review presents the different formats and conditions for the use of lectins in affinity chromatography and in solid phase extraction, including their use in dispersive mode, along with the recent progress made on either commercial or home-made lectin-based affinity sorbents, which can lead to a fast and automated glycosylation analysis.

Fc gamma receptor IIIb binding of individual antibody proteoforms resolved by affinity chromatography-mass spectrometry.

Abstract: The crystallizable fragment (Fc) of immunoglobulin G (IgG) activates key immunological responses by interacting with Fc gamma receptors (FcɣR). FcɣRIIIb contributes to neutrophil activation and is ...

Monoclonal antibody against l-lectin module of SraP blocks adhesion and protects mice against Staphylococcus aureus challenge.

Abstract: Background/purpose SraP is a serine-rich repeat protein (SRRP) from Staphylococcus aureus that binds to sialylated receptors to promote bacterial adhesion to and invasion into host epithelial cells, mediated by the l -lectin module of its ligand-binding region. Methods The sequence encoding the L -lectin module of SraP was inserted into pET28a plasmid, and the recombinant protein was purified by His label affinity chromatography. A monoclonal antibody (mAb) against the l -lectin module was obtained and confirmed by enzyme-linked immunosorbent assay and western blotting. The effect of the mAb on S. aureus adhesion and invasion was assessed in A549 cells and mice subjected to S. aureus challenge. Results We successfully obtained a mAb against the l -lectin module of SraP. Pre-incubation with the mAb dramatically inhibited the bacteria's ability to adhere to and invade A549 cells. Moreover, mice administered mAb through tail vein injection had significantly fewer bacteria in the blood. Conclusion The anti-SraPL-Lectin mAb significantly reduced the adherence and invasion of S. aureus to host cells. This study lays the foundation for the future development of the l -lectin module of SraP as a target for the prevention and treatment of S. aureus infection. Our findings suggest that specific subdomains of SRRPs may represent potential antibacterial drug targets for intervention.

Glutathione S-transferase: Purification and Characterization from Quail ( Coturnix coturnix japonica ) Liver and the Impact of Some Metal Ions on Enzyme Activity

Abstract: The objective of this study was to examine the inhibitory effect of some heavy metals on the glutathione S-transferase (GST) enzyme, which is one of the major enzymes of glutathione metabolism purified from quail liver tissues. The quail liver GST enzyme was purified with 15.86 EU/mg specific activity, in a yield of 12.36% and 46.1 purification fold by ammonium sulfate precipitation and glutathione-agarose affinity chromatography. The molecular weight of subunits of the enzyme and the purity were determined by SDS-PAGE. In the characterization studies, the optimum pH of the GST enzyme was determined to be pH = 8.0 in Tris/HCl buffer. Optimum ionic strength was determined to be 140 mM in Tris/HCI buffer. Stable pH was found to be pH = 8.5 in Tris/HCl buffer. Optimum temperature was found to be 50 °C. KM and Vmax values for substrates 1-chloro-2,4-dinitrobenzene (CDNB) and GSH of the enzyme were determined to be KM 0.048 mM Vmax 0.479 EU/mL and KM 0.114 mM Vmax 0.672 EU/mL, respectively. In vitro inhibition effects of metal ions, including Ag+, Ni2+, Cd2+, Fe2+, Pb2+, Co2+ Zn2+, and Al3+, were investigated on the GST enzyme activity. The results showed that Ag+, Cd2+, Ni2+ Zn2+, and Al3+ metal ions inhibited GST enzyme (IC50 values 0.239, 0.250, 0.265, 0.320, 0.594 mM, respectively), while Fe2+, Pb2+, and Co2+ metal ions activated the enzyme. Finally, Ki values and inhibition types for these substances were determined by Lineweaver-Burk graphs.

Affinity Chromatography: A Powerful Tool in Drug Discovery for Investigating Ligand/membrane Protein Interactions

Abstract: Affinity chromatography is an often overlooked chromatographic technique regarding receptor/ligand studies. Indeed, biologists have a tendency to focus on more recent biophysical techniques such as...

Washing with alkaline solutions in protein A purification improves physicochemical properties of monoclonal antibodies

Abstract: Protein A affinity chromatography has been widely used for both laboratory scale purification and commercial manufacturing of monoclonal antibodies and Fc-fusion proteins. Protein A purification is specific and efficient. However, there still remain several issues to be addressed, such as incomplete clearance of impurities including host cell proteins, DNA, aggregates, etc. In addition, the effects of wash buffers in protein A purification on the physicochemical characteristics of antibodies have yet to be fully understood. Here we found a new purification protocol for monoclonal antibodies that can improve physicochemical properties of monoclonal antibodies simply by inserting an additional wash step with a basic buffer after the capture step to the conventional protein A purification. The effects of the alkaline wash on monoclonal antibodies were investigated in terms of physicochemical characteristics, yields, and impurity clearance. The simple insertion of an alkaline wash step resulted in protection of antibodies from irreversible aggregation, reduction in free thiols and impurities, an improvement in colloidal and storage stability, and enhanced yields. This new procedure is widely applicable to protein A affinity chromatography of monoclonal antibodies.

Cloning, Expression and One-Step Purification of a Novel IP-10-(anti-HER2 scFv) Fusion Protein in Escherichia coli

Abstract: Breast cancer is the most common cancer in women, worldwide. The correlation between breast cancer malignancy and human epidermal growth factor 2 (HER2) expression leads to application of monoclonal antibodies against HER2 in HER2-overexpressing breast cancers. Variable fragments of light and heavy chains of monoclonal antibodies are linked in single chain variable fragment (scFv). Herein, IP-10 chemokine is conjugated to scFv against HER2 and thus CD8+T cells can chemoattract to HER2-overexpressing tumors. IP-10-(anti-HER2 scFv) gene was cloned in pET22-b (+) and successful expression of the fusion protein in E. coli host was confirmed by detecting a 39 kDa band in Western blotting. The highest fusion protein expression in Origami (DE3), BL21 (DE3) and SHuffle® were obtained 4 h after 0.1, 0.5 and 0.1 mM IPTG induction at 37, 37 and 30 °C, respectively. It was also demonstrated that the most solubility of the fusion protein is obtained at 25 °C in all the three examined strains. The highest soluble form of the fusion protein was expressed in SHuffle®. However, due to low amount of the fusion protein expressed in SHuffle®, we focused on its expression in Origami (DE3). Finally, purification of the fusion protein using Ni–NTA affinity chromatography under native, denaturing and hybrid conditions was investigated. Purification under hybrid condition caused the most purity of the fusion protein. This study opens up the avenue of exploring the use of IP-10-(anti-HER2 scFv) as a potential treatment for HER2-overexpresing tumors or as an adjuvant in combination with HER2-based vaccine.