Affinity Chromatography Media Research Articles

Screening and isolation of quorum sensing inhibitors of Pseudomonas aeruginosa from Phellodendron amurense extracts using bio-affinity chromatography.

Drug-resistant bacterial infections pose a significant challenge in the field of bacterial disease treatment. Finding new antibacterial pathways and targets to combat drug-resistant bacteria is crucial. The bacterial quorum sensing (QS) system regulates the expression of bacterial virulence factors. Inhibiting bacterial QS and reducing bacterial virulence can achieve antibacterial therapeutic effects, making QS inhibition an effective strategy to control bacterial pathogenicity. This article mainly focused on the PqsA protein in the QS system of Pseudomonas aeruginosa. An affinity chromatography medium was developed using the SpyTag/SpyCatcher heteropeptide bond system. Berberine, which can interact with the PqsA target, was screened from Phellodendron amurense by affinity chromatography. We characterized its structure, verified its inhibitory activity on P. aeruginosa, and preliminarily analyzed its mechanism using molecular docking technology. This method can also be widely applied to the immobilization of various protein targets and the effective screening of active substances.

Recovery of human serum albumin by dual-mode chromatography from the waste stream of Cohn fraction V supernatant.

Plasma fractionation industry is by far the largest protein pharmaceutical provider, but there are still some plasma components in its industrial waste liquid that have not been utilized. This study aimed to develop a simple and efficient method for plasma protein recovery from Cohn fraction V supernatant (FVS), an effluent containing about 40% ethanol. A new affinity chromatography medium was synthesized with a fatty acid ligand. When the medium was applied to recovery of human serum albumin (HSA) from FVS at physiological pH7.4, the process was unsuccessful due to substantial decrease in capacity in the presence of high ethanol concentration. Nevertheless, change of pH from 7.4 to 4.2 emerged an improved adsorption capacity. The carboxyl group of the ligand began to act as cationic ion exchange role. Both HSA and α2HS-glycoprotein were adsorbed by the column, but α2HS-glycoprotein could be eluted by increasing pH from 4.2 to 7.4, while HSA was retained by the column and could only be eluted by addition of fatty acid. Therefore, the adsorption of albumin under pH 4.2 is charge-induced affinity adsorption, not simple ion exchange. The so-called dual-mode adsorption depends not only on the chromatographic medium but also on the separated object and environment. HPSEC showed that the purity of recovered HSA was greater than 98%. Circular dichroism and fluorescence spectra were consistent with that of the commercial product. Furthermore, the measurement by isothermal titration calorimetry showed that the separated HSA still maintained the binding activities with the ligands of warfarin and naproxen. It is therefore possible to directly recover high-purity and high-quality human serum albumin from the effluent of plasma fractionation industry by one-step chromatography.

Improved protein purification system based on C-terminal cleavage of Npu DnaE split intein.

A purification system was constructed with the N-segment of the Npu DnaE split intein as an affinity ligand immobilized onto an epoxy-activated medium and the C-segment used as the cleavable tag fusing target protein. The affinity properties of C-tagged proteins adsorbed on IN affinity chromatography medium were studied with GFP as a model target protein. The saturated adsorption capacity and dynamic adsorption capacity reached 51.9-21.0mgmL-1, respectively. With this system, two model proteins, GFP and alcohol dehydrogenase (ADH), has been successfully taglessly purified with regulation of Zn2+ and DTT. The yield, purification factor and purity of purified tagless GFP reached 39, 11.7 and 97%, respectively; while these values for purified tagless ADH were 38.2, 6.8 and 91%, respectively. These results showed that the system for Npu DnaE split intein-mediated affinity adsorption and in situ cleavage is a potential platform for recombinant protein production.

Improved adsorption reactions, kinetics and stability for model and therapeutic proteins immobilised\xa0on affinity resins

Protein adsorption on solid state media is important for the industrial affinity chromatography of biotherapeutics and for preparing materials for self-interaction chromatography where fundamental protein solution thermodynamic properties are measured. The adsorption of three model proteins (lysozyme, catalase and BSA) and two antibodies (a monoclonal and a polyclonal antibody) have been investigated on commercial affinity chromatography media with different surface functionalities (Formyl, Tresyl and Amino). Both the extent of protein immobilised (mg protein/ml media) and the reaction kinetics are reported for a range of reaction conditions, including pH, differing buffers as well as the presence of secondary reactants (glutaraldehyde, sodium cyanoborohydride, EDC and NHS). Compared to the reaction conditions recommended by manufacturers as well as those reported in previous published work, significant increases in the extent of protein immobilisation and reaction kinetics are reported here. The addition of glutaraldehyde or sodium cyanoborohydride was found to be especially effective even when not directly needed for the adsorption to happen. For mAb and pIgG, immobilisation levels of 50 and 31 mg of protein/ml of resin respectively were achieved, which are 100% or more than previously reported. Enhanced levels were achieved for lysozyme of 120 mg/ml with very rapid reaction kinetics (< 1 h) with sodium cyanoborohydride. It can be concluded that specific chromatography resins with Tresyl activated support offered enhanced levels of protein immobilisation due to their ability to react to form amine or thio-ether linkages with proteins. Additionally, glutaraldehyde can result in higher immobilisation levels whilst it can also accelerate immobilisation reaction kinetics.

Heparin‐Immobilized Polymeric Monolithic Column with Submicron Skeletons and Well‐Defined Macropores for Highly Efficient Purification of Enterovirus 71

AbstractIn this study, glycidyl methacrylate‐based polymerized high internal phase emulsion monolithic column (polyHIPE) with submicron skeletons and well‐defined macropores is prepared and functionalized by immobilizing heparin to obtain a novel affinity chromatographic separation medium for effective purification of Enterovirus 71 (EV71). The pore structure of the obtained monoliths differs significantly from conventional polyHIPE monoliths. The prepared polyHIPE presents well‐defined 3D network skeletons of 0.2–0.5 µm, interconnected macropores of 0.5–2 µm, large specific surface area (47–61 m2 g−1), and excellent permeability (k = 1.0 × 10−13 m2). Further, successful immobilization of heparin is proved by FTIR and XPS and the monoliths display superiority in the purification of EV71 from culture supernatant. The final recovery and binding capacity of EV71 are calculated to be 60% and 152.8 ng per column, respectively. SDS‐PAGE gel electrophoresis and TEM analysis indicate a high efficiency in the removal of impurities from culture supernatant of EV71. This, together with easy fabrication, makes polyHIPE monolith a promising alternative to commercially available monolithic supports for virus purification.

Characterization of a P-selectin-binding moiety from Bupleurum chinense polysaccharide and its antagonistic effect against P-selectin-mediated function

P-selectin is a promising therapeutic target for acute inflammation-related diseases, and interest has been growing in the search for high-affinity glycoconjugate ligands that can target the initial P-selectin-mediated recruitment of neutrophils to the site of inflammation. In our previous study, we isolated a water-soluble polysaccharide (BCPS) from Bupleurum chinense and showed that it exhibits anti-inflammatory effect by antagonizing P-selectin-mediated adhesion of HL-60 cells to CHO-P cells. In this study, we prepared a P-selectin-based affinity chromatography medium and used it to purify the P-selectin-binding moiety of BCPS. The purified P-selectin-binding moiety of BCPS, designated as BCPS-m, was mainly composed of arabinose, galactose and glucose, and had a relative molecular weight of 3600 Da. The backbone of BCPS-m was composed of 1,5-linked arabinose, 1,4-linked and 1,4,6-linked glucose, and with branched 1-linked glucose or galactose terminal. BCPS-m could disrupt the P-selectin-mediated binding of HL-60 cells to CHO-P cells (CHO cells that stably expressed an exogenous P-selectin). It also blocked the interaction between P-selectin and its physiological ligand PSGL-1 significantly, resulting in much greater reduction (77%) in P-selectin-PSGL-1 binding than that caused by BCPS (35%). The data suggested that BCPS-m could be the key P-selectin-binding moiety of BCPS, and that it may be a better P-selectin antagonist than BCPS.

Front Cover: Enhanced binding by dextran-grafting to Protein A affinity chromatographic media

Front Cover: Enhanced binding by dextran-grafting to Protein A affinity chromatographic media

Enhanced binding by dextran-grafting to Protein A affinity chromatographic media.

Dextran-grafted Protein A affinity chromatographic medium was prepared by grafting dextran to agarose-based matrix, followed by epoxy-activation and Protein A coupling site-directed to sulfhydryl groups of cysteine molecules. An enhancement of both the binding performance and the stability was achieved for this dextran-grafted Protein A chromatographic medium. Its dynamic binding capacity was 61mg immunoglobulin G/mL suction-dried gel, increased by 24% compared with that of the non-grafted medium. The binding capacity of dextran-grafted medium decreased about 7% after 40 cleaning-in-place cycles, much lower than that of the non-grafted medium as decreased about 15%. Confocal laser scanning microscopy results showed that immunoglobulin G was bound to both the outside and the inside of dextran-grafted medium faster than that of non-grafted one. Atomic force microscopy showed that this dextran-grafted Protein A medium had much rougher surface with a vertical coordinate range of ±80nm, while that of non-grafted one was ±10nm. Grafted dextran provided a more stereo surface morphology and immunoglobulin G molecules were more easily to be bound. This high-performance dextran-grafted Protein A affinity chromatographic medium has promising applications in large-scale antibody purification.

Choosing the right protein A affinity chromatography media can remove aggregates efficiently.

Protein A chromatography (PAC) is commonly used as an efficient capture step in monoclonal antibody (mAb) separation processes. Usually dynamic binding capacity is used for choosing the right PAC. However, if aggregates can be efficiently removed during elution, it can make the following polishing steps easier. In this study a method for choosing the right PAC media in terms of mAb aggregate removal is proposed. Linear pH gradient elution experiments of two different mAbs on various PAC columns are carried out, where the elution behavior of aggregates as well as the monomer is measured. Aggregates of one mAb are more strongly retained compared with the mAb monomer. Another mAb showed different elution behavior, where the aggregates are eluted as both the weakly and strongly retained peaks. In order to remove the two types of aggregates by stepwise elution two protocols are tested. The first protocol A consisted of the sample loading, the wash with the equilibration buffer and the low pH elution. The wash stage of the second protocol B included the wash with 1.0 M arginine. No detectable peaks are observed during the wash stage of protocol A whereas significant peaks are monitored during the arginine wash of protocol B. One of the PAC columns showed a smaller peak during the arginine wash. In addition, both aggregate removal and monomer yield are higher with protocol B compared with the other PAC columns. This method is found to be useful for choosing the right PAC column.

Efficient fabrication of high‐capacity immobilized metal ion affinity chromatographicmedia: The role of the dextran‐grafting process and its manipulation

J. Sep. Sci. 2016, 39, 1130–1136 DOI: 10.1002/jssc.201501291 A novel high-capacity Ni2+ immobilized metal ion affinity chromatographic medium was prepared through dextran-grafting process. The dextran-grafting degree of the media had a great effect on His-tagged protein binding performance. The maximum binding capacity was obtained as a certain thickness of dextran was grafted.

Preparation and properties of new nickel ion affinity chromatography medium

With the chloromethylated crosslinking polystyrene microspheres (chlorine ball) as the matrices, N,N⁃dimethylformamide (DMF) as the solvent, activated by N,N⁃diisopropyleth⁃ ylamine (DIEA), the synthesized Lys(Boc) ⁃OEt and ethyl chloroacetate were coupled to the matrices. After hydrolysis, the carboxyl group was released and chelated with Ni, and a new nickel ion affinity chromatographic medium was prepared. The affinity medium was applied to purify the His⁃Lys⁃Tyr and Phe⁃His⁃Thr tri⁃peptides. The results showed that the peptides con⁃ taining histidine were retained on the affinity medium and separated with other substances.

Development of a novel affinity chromatography resin for platform purification of lambda fabs.

Antigen-binding fragments (Fabs) are novel formats in the growing pipeline of biotherapeutics. Sharing similar features to monoclonal antibodies (mAbs) with regard to expression, Fabs are considered as unchallenging for upstream development. Yet for downstream processing, the mature mAb downstream purification platform is not directly applicable. New approaches need to be found to achieve a lean purification process that maintains quality, productivity, and timelines while being generically applicable independent of the expression system. In a successful collaboration, BAC BV, GE Healthcare, and Novartis Pharma AG have developed a new affinity chromatography medium (resin) suitable to support cGMP manufacturing of lambda Fabs. We show that using this novel chromatography medium for the capture step, a purification platform for lambda Fabs can be established.

Expression and fast preparation of biologically active recombinant human coagulation factor VII in CHO-K1 cells

Human coagulation factor VII (FVII) plays an important role in the blood coagulation process and exists in micro amounts in human plasma; therefore, any attempt at the large-scale production of FVII in significant quantities is challenging. The purpose of this study was to express and obtain biologically active recombinant FVII (rFVII) from Chinese hamster ovary K1 (CHO-K1) cells. The full-length FVII cDNA was isolated from a HepG2 cell line and then subcloned in pcDNA3.1 to construct an expression vector, pcDNA-FVII. CHO-K1 cells were transfected with 1 µg pcDNA-FVII. The cell line that stably expressed secretory FVII was screened using 900 µg/mL G418. The FVII copy number in CHO-K1 cells was detected by quantitative polymerase chain reaction (qPCR). The rFVII was purified in ligand affinity chromatography medium. The purified protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The biological activity of the purified FVII protein was determined by a prothrombin time assay. Three cell lines that permanently expressed rFVII were screened. The qPCR results demonstrated that each CHO-K1 cell harbored two FVII DNA copies. The SDS-PAGE and Western blot analysis showed that the purified protein was about 50 kDa. The purity of the target protein was 95%. The prothrombin time assay indicated that the FVII-specific activity of rFVII was 2573 ± 75 IU/mg. This method enabled the fast preparation of high-purity rFVII from CHO-K1 cells, and the purified protein had good biological activity.

Large-scale preparation and characterization of zinc-substituted hydroxyapatite for metal affinity chromatography

The preparation method for the surface zinc-modified hydroxyapatite particles that we developed as immobilized metal ion affinity chromatography medium, required decantation process to remove the excess zinc ion. Therefore, large-scale production was sometimes difficult because of this process. In this report, we describe our improved method to synthesize zinc-substituted hydroxyapatite (CaZnHAp) particles and their further chromatographic properties. This method is simple, easy to scale up, and does not require decantation process. CaZnHAp was synthesized from Ca(OH)2, H3PO4, and ZnO by a wet method with spray-dryer. This method proved suitable for large-scale production and the quantity of substituted zinc ions was stable without decantation process, as indicated by X-ray photoelectron spectroscopic analysis. The analysis showed that Zn ions were located on the surface and further inside CaZnHAp, while located on only the surface of the conventional material, therefore, CaZnHAp showed stronger adsorbability and durability. Chromatographic separation of histidine oligomers (monomer, dimer, tetramer, and hexamer of histidine) and histidine-tagged protein from CaZnHAp was compared with the results obtained with other materials. These peptides and proteins were more strongly retained by CaZnHAp than other materials and could be separated in one step. This preparation method can serve to obtain hydroxyapatite with stronger affinity.

Characterization of Conformational Changes and Protein-Protein Interactions of Rod Photoreceptor Phosphodiesterase (PDE6)

As the central effector of visual transduction, the regulation of photoreceptor phosphodiesterase (PDE6) is controlled by both allosteric mechanisms and extrinsic binding partners. However, the conformational changes and interactions of PDE6 with known interacting proteins are poorly understood. Using a fluorescence detection system for the analytical ultracentrifuge, we examined allosteric changes in PDE6 structure and protein-protein interactions with its inhibitory γ-subunit, the prenyl-binding protein (PrBP/δ), and activated transducin. In solution, the PDE6 catalytic dimer (Pαβ) exhibits a more asymmetric shape (axial ratio of 6.6) than reported previously. The inhibitory Pγ subunit behaves as an intrinsically disordered protein in solution but binds with high affinity to the catalytic dimer to reconstitute the holoenzyme without a detectable change in shape. Whereas the closely related PDE5 homodimer undergoes a significant change in its sedimentation properties upon cGMP binding to its regulatory cGMP binding site, no such change was detected upon ligand binding to the PDE6 catalytic dimer. However, when Pαβ was reconstituted with Pγ truncation mutants lacking the C-terminal inhibitory region, cGMP-dependent allosteric changes were observed. PrBP/δ bound to the PDE6 holoenzyme with high affinity (K(D) = 6.2 nm) and induced elongation of the protein complex. Binding of activated transducin to PDE6 holoenzyme resulted in a concentration-dependent increase in the sedimentation coefficient, reflecting a dynamic equilibrium between transducin and PDE6. We conclude that allosteric regulation of PDE6 is more complex than for PDE5 and is dependent on interactions of regions of Pγ with the catalytic dimer.

Importance of pH and disulfide bridges on the structural and binding properties of human α 1-acid glycoprotein

Human α 1-acid glycoprotein (AGP) is an acute phase plasma glycoprotein containing two disulfide bridges. As a member of the lipocalin superfamily, it binds and transports several basic and neutral ligands, but a number of other activities have also been described. Thanks to its binding properties, AGP is also a good candidate for the development of biosensors and affinity chromatography media, and in this context detailed structural information is needed. The structural properties of AGP at different p 2Hs and under reducing conditions were analysed by FT-IR spectroscopy. The obtained data indicate that AGP, when denatured, does not aggregate at neutral or basic p 2Hs whilst it does at acidic p 2Hs. Under reducing conditions the protein is remarkably less thermostable than its oxidized counterpart and presents an enhanced tendency to aggregate, even at neutral p 2H. A heat-induced molten globule-like state (MG) was detected at 55 °C at p 2H 7.4 and 5.5. At p 2H 4.5 the MG occurred at 45 °C with an onset of formation at 40 °C. The MG was not observed under reducing conditions. A lower affinity of chlorpromazine and progesterone for the MG formed at p 2H 4.5 and 40 °C was observed, suggesting that ligand(s) may be released near the negative surfaces of biological membranes. Furthermore, the reduced AGP displays an enhanced affinity for progesterone, indicating the importance of disulfide bonds for the binding capacity of AGP.

Phosphorylcholine (PC)-bonded Protein A Affinity Chromatographic Medium for High-Throughput Purification with Reduced Non-specific Protein Adsorption

A protein A affinity chromatographic medium based on porous silica modified with phosphorylcholine (PC) groups and amino groups (PNSP) was synthesized. The PC groups functioned as suppressors of non-specific protein adsorption. Recombinant protein A was bound to the amino groups on PNSP with a glutaraldehyde used as a spacer (PNSP-PA). The PC groups and amino groups were immobilized on porous-silica particles using two silane coupling reagents, PC-bound silane, and 3-aminopropyltrimethoxysilane. After optimizing various factors in the synthetic process, the resultant protein A medium showed improvements in non-specific protein adsorption, dynamic binding capacity, and chemical stability under basic conditions compared with conventional protein A affinity media.

A defensin with highly potent antipathogenic activities from the seeds of purple pole bean

A 5443 Da peptide with sequence homology to defensins was purified from purple pole beans (Phaseolus vulgaris cv. 'Extra-long Purple Pole bean'). This peptide was isolated by adsorption on an affinity chromatographic medium Affi-Gel Blue gel and ion-exchange chromatographic media SP-Sepharose (sulfopropyl-Sepharose) and Mono S and by gel filtration on Superdex peptide. The peptide inhibited mycelial growth in Mycosphaerella arachidicola, Helminthosporium maydis, Fusarium oxysporum, Verticillium dahliae, Rhizoctonia solani, Candida albicans and Setosphaeria turcica with an IC50 of 0.8, 0.9, 2.3, 3.2, 4.3, 4.8 and 9.8 microM respectively. Its antifungal potency was higher than that of the plant defensin coccinin (IC50>50 microM). It induced membrane permeabilization in C. albicans as evidenced by SYTOX Green uptake, but did not affect erythrocyte membrane permeability. It inhibited growth in M. arachidicola by inducing chitin accumulation at hyphal tips as was shown by Congo Red staining. The antifungal activity was pH stable and thermostable. The peptide inhibited the proliferation of hepatoma (HepG2), breast cancer (MCF7), colon cancer (HT29) and cervical cancer (SiHa) cells but not that of human embryonic liver (WRL68) cells. Its anti-HepG2 activity (IC50=4.1+/-0.8 microM, n=3) was higher than that of another plant defensin, gymnin (IC50>50 microM). Its anti-MCF7 activity (IC50=8.3+/-0.3 microM, n=3) was similar to that of other plant defensins. It reduced the activity of HIV-1 reverse transcriptase with an IC50 of 0.5+/-0.1 microM, n=3, much more potently than other plant defensins (IC50>40 microM). There is the possibility of using the purple pole bean defensin for producing antifungal drugs and/or transgenic plants with fungal resistance.

Resin optimization for affinity chromatography media

Resin optimization for affinity chromatography media

Single-step purification of recombinant green fluorescent protein on expanded beds of immobilized metal affinity chromatography media

Abstract Immobilized metal ion affinity chromatography (IMAC) in expanded bed mode is used for purifying recombinant green fluorescent protein (GFP) overexpressed in Escherichia coli. The purification is carried out on two different matrices, i.e. Ni2+ Streamline™ and Ni2+ cross-linked alginate beads. The binding isotherms to both IMAC media followed the Langmuir model. The maximum binding capacity (qmax) of Ni2+ Streamline™ and Ni2+ cross-linked alginate for the GFP was 1,42,860 FU ml−1 and 18,000 FU ml−1, respectively. The expanded bed column chromatography using Ni2+ Streamline™ gave 2.7-fold purification with 89% of GFP recovery, while Ni2+ alginate gave 3.1-fold purification with 91% of GFP recovery. SDS-PAGE of purified GFP in both cases showed single band. The results obtained in the expanded bed chromatography are compared with those obtained in packed bed chromatography.