Protein Chromatography Research Articles

Coating of nanoparticles on cryogel surface and subsequent double-modification for enhanced ion-exchange capacity of protein

A novel composite cryogel monolith was developed by coating poly(glycidyl methacrylate) nanoparticles (NPs) onto the pore wall surface of poly(acrylamide) cryogel. The NPs-coated column was double-modified with poly(ethylenimine) (PEI) and diethylaminoethyl in sequence. Scanning electron microscopy revealed the dense coating of the NPs on the cryogel surface, but the NPs-coating did not result in distinct changes of the column porosity and permeability. The rough pore wall surface and extended polymer chains offered more binding sites, so the dynamic binding capacity of the composite cryogel bed for bovine serum albumin reached 11.7mg/mL bed volume at a flow rate of 6cm/min, which was 4.2 times higher than that of the cryogel bed modified with PEI without coating NPs (2.8mg/mL). The binding capacity as well as column efficiency decreased only slightly with increasing flow rate from 0.6 to 12cm/min. The results indicated that the strategy of NPs-coating incorporating with double ion-exchanger modifications is promising for enhancing cryogel capacities, and the novel material would be useful for high-speed protein chromatography.

A microporous walled micro-capillary film module for cation-exchange protein chromatography

Opportunities exist in preparative chromatography for alternative chromatography media that possess high binding capacity and throughput, but are also economically feasible for single use disposability and avoid column packing. An ion-exchange functionalised, microporous walled micro-capillary film (MMCF), has been developed as a module for cation-exchange separation of proteins. A MMCF module has been operated on a standard AKTA chromatography system at pressures up to 1.5MPa and superficial flow velocities up to 54,000cmh−1. The dynamic binding capacity of the MMCF module at 10% breakthrough was 13.8mglysozyme/ml adsorbent volume, which is comparable to the capacity of current commercial adsorbents. Frontal analysis studies using a mixture of lysozyme and bovine serum albumin (BSA) have shown that lysozyme can be isolated free of BSA to the limit of detection of the SDS gel assay used. 98.8% of the total sample eluted was the target protein lysozyme with only 1.2% BSA impurity. MMCF may thus be a viable chromatographic medium for preparative protein chromatography.

Proteomic mapping of the lung vascular endothelial cell surface in Schistosoma bovis-infected hamsters.

To identify the changes induced by schistosomula larvae of Schistosoma bovis in the proteome of the pulmonary vasculature of the host, we compared the proteins expressed on the vascular endothelium of the lungs of non-infected and infected hamsters over 10 and 20 days. Mass spectrometry analysis (LC-MS/MS) of the proteins isolated from the vascular endothelium resulted in the identification of a total of 459 non-redundant proteins in the lung of infected and non-infected hamsters. The proteins identified are classified according to their biological function and cellular location, further analysing the differences in lung vascular proteomes between non-infected and S. bovis-infected hamsters. This work provides the first data on the vascular surface proteome of the lung after S. bovis infection and identifies some of the changes induced in it during infection suggesting the possible involvement of these proteins during parasite infection.

Comparison of DNA-Hydrolyzing Antibodies from the Cerebrospinal Fluid and Serum of Patients with Multiple Sclerosis

It was found that high-affinity anti-DNA antibodies were one of the major components of the intrathecal IgG response in multiple sclerosis (MS) patients [Williamson et al., PNAS, 2001]. Recently we have shown that IgGs from the sera of MS patients are active in the hydrolysis of DNA. Here we have shown, for the first time, that average concentration of total proteins (132-fold), total IgGs (194-fold) and anti-DNA antibodies (200-fold) in the sera is significantly higher than that in the cerebrospinal fluid (CSF) of fifteen MS patients. The relative activities of total protein from sera and CSFs varied remarkably from patient to patient. It was surprising that the specific DNase activity of the total protein of CSF reparations were 198-fold higher than the serum ones. Electrophoretically and immunologically homogeneous IgGs were obtained by sequential affinity chromatography of the CSF proteins on protein G-Sepharose and FPLC gel filtration. We present first evidence showing that IgGs from CSF not only bind but efficiently hydrolyze DNA and that average specific DNase activity of homogeneous antibodies from CSF is unpredictably ∼49-fold higher than that from the sera of the same MS patients. Some possible reasons of these findings are discussed. We suggest that DNase IgGs of CSF may promote important neuropathologic mechanisms in this chronic inflammatory disorder and MS pathogenesis development.

Biomimetic design of affinity peptide ligands for human IgG based on protein A-IgG complex

Staphylococcal protein A (SpA) has been widely used as an affinity ligand for the purification of immunoglobulin G (IgG). Based on the affinity motif of SpA, we have herein developed a biomimetic design strategy for affinity peptide ligands of IgG. First, according to the distribution of the six hot spots of the SpA affinity motif determined previously, the number of residues that should be inserted into between the hot spots was determined. Cysteine was introduced as one of the middle inserted residues of the peptide for later immobilization. Then, amino acid location was performed to identify other amino acid residues for insertion, leading to the construction of a peptide library. The library was screened by using different molecular simulation protocols, resulting in the selection of 15 peptide candidates. Thereafter, molecular dynamics simulations were performed to validate the dynamics of the affinity interactions between the candidates and IgG, and 14 of them were found to keep high affinities. Finally, the affinity and specificity of the top one ligand FYWHCLDE were exemplified by protein chromatography and IgG purification. The results indicate that the design strategy was successful and the affinity peptide ligand for IgG is promising for application in antibody purifications.

Escherichia coli kduD encodes an oxidoreductase that converts both sugar and steroid substrates

A previously unidentified oxidoreductase from Escherichia coli catalyzes the regioselective reduction of eukaryotic steroid hormone 11-deoxycorticosterone (11-DOC) to the valuable bioactive product 4-pregnen-20,21-diol-3-one. In nature, a reduction of C-20 carbonyl of C21 steroids is catalyzed by diverse NAD(P)H-dependent oxidoreductases. Enzymes that possess 20-ketosteroid reductase activity, however, have never before been described in E. coli. Our present study aimed to identify and characterize the E. coli enzyme which possesses 20-ketosteroid reductase activity against eukaryotic steroid hormone 11-DOC. We partially purified the enzyme from E. coli DH5α using protein chromatography techniques. Mass spectrometry revealed the presence of three NADH-specific oxidoreductases in the sample. The genes encoding these oxidoreductases were cloned and overexpressed in E. coli UT5600 (DE3). Only the overexpression of 2-dehydro-3-deoxy-D-gluconate 5-dehydrogenase (KduD) encoded by kduD gene enabled the whole-cell biotransformation of 11-DOC. A 6xHis-tagged version of KduD was purified to homogeneity and found to reduce several eukaryotic steroid hormones and catalyze the conversion of novel sugar substrates. KduD from E. coli is therefore a promiscuous enzyme that has a predicted role in sugar conversion in vivo but can be used for the production of valuable bioactive 20-hydroxysteroids.

Evaluation of steric exclusion chromatography on cryogel column for the separation of serum proteins

Steric exclusion chromatography (SXC) is a new mode of protein chromatography, in which large proteins are retained on hydrophilic stationary phase surface due to the steric exclusion of polyethylene glycol (PEG) in the mobile phase, and thereafter the retained proteins can be eluted by reducing PEG concentration. In this work, SXC was evaluated on a polyacrylamide cryogel monolith. Microscopic observation of γ-globulin precipitates on the gel surface in SXC was reported for the first time. Due to the compact packing of protein precipitates on the stationary phase surface, the dynamic retention capacity of the cryogel monolith for γ-globulin reached 20mg/mL bed volume, much higher than those of cryogel beds in adsorption-based chromatography. The effect of molecular weight and concentration of PEG, solution pH and salt concentration on protein retention capacity was in agreement with the earlier work on SXC. Because the cryogel monoliths with interconnected macropores (10–100μm) allow much easy flow-through of viscous PEG buffer, the SXC can be operated at low back pressure. Hence, the cryogel monoliths are more suitable for SXC than other monoliths of narrow pores reported previously. In the separation of bovine serum proteins, albumin was recovered in the breakthrough fraction with high purity, and globulin was over eight times concentrated in the elution pool. This work has, thus, demonstrated the rapid serum protein separation and concentration by SXC on the cryogel monolith columns.

Antioxidant, Antibacterial, and Cytoprotective Activity of Agathi Leaf Protein

In the present study a protein termed agathi leaf protein (ALP) from Sesbania grandiflora Linn. (agathi) leaves was isolated after successive precipitation with 65% ammonium sulphate followed by purification on Sephadex G 75. The column chromatography of the crude protein resulted in four peaks of which Peak I (P I) showed maximum inhibition activity against hydroxyl radical. SDS-PAGE analysis of P I indicated that the molecular weight of the protein is ≈29 kDa. The purity of the protein was 98.4% as determined by RP-HPLC and showed a single peak with a retention time of 19.9 min. ALP was able to reduce oxidative damage by scavenging lipid peroxidation against erythrocyte ghost (85.50 ± 6.25%), linolenic acid (87.67 ± 3.14%) at 4.33 μM, ABTS anion (88 ± 3.22%), and DNA damage (83 ± 4.20%) at 3.44 μM in a dose-dependent manner. The purified protein offered significant protection to lymphocyte (72% at 30 min) induced damage by t-BOOH. In addition, ALP showed strong antibacterial activity against Pseudomonas aeruginosa (20 ± 3.64 mm) and Staphylococcus aureus (19 ± 1.53 mm) at 200 μg/mL. The safety assessment showed that ALP does not induce cytotoxicity towards human lymphocyte at the tested concentration of 0.8 mg/mL.

Reversible Immobilization of Chelating Affinity Surfactants on Reversed Phase Adsorbents for Protein and Peptide Separations under Metal Affinity Chromatography

Alkyl-bound silica was modified using chelating surfactants and the resulting adsorbent was used in immobilized metal affinity chromatography of proteins and peptides. Brij-76, a non-ionic amphiphilic surfactant with an alkyl moiety and an ethylene oxide chain, was reversible adsorbed to alkyl silica (C18). The hydroxyl group at the end of the ethylene oxide chain was chemically modified previously with an iminodiacetate functionality as chelating agent of transitional metal ions. Cu(II) was studied as immobilized ion for the adsorption of peptides and proteins. Three chromatographic supports were prepared having different Cu(II) capacities. For a low Cu(II) capacity case, the generated adsorbent behaved as a controlled access media preventing the adsorption of large molecular weight proteins, such as BSA, while small peptides, such as Angiotensin III, or amino acids could be retained. For a medium and high Cu(II) capacity, the synthesized adsorbent no longer behaved as a controlling access media and all molecules in this study, either large or small, were retained by the immobilized ion. Nonetheless, most of the BSA was strongly retained by the system and a pH change did not remove any of the adsorbed BSA while the small molecules were removed by the same pH change.

3P049 Multimodal chromatography of proteins in arginine solutions(01C. Protein: Property,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))

3P049 Multimodal chromatography of proteins in arginine solutions(01C. Protein: Property,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))

A novel gigaporous GSH affinity medium for high-speed affinity chromatography of GST-tagged proteins

Novel GSH-AP (phenoxyl agarose coated gigaporous polystyrene, Agap-co-PSt) microspheres were successfully prepared by introducing GSH ligand into hydrophilic AP microspheres pre-activated with 1,4-butanediol diglycidyl ether. The gigaporous structure and chromatographic properties of GSH-AP medium were evaluated and compared with commercial GSH Sepharose FF (GSH-FF) medium. The macropores (100–500nm) of gigaporous PSt microspheres were well maintained after coating with agarose and functionalized with GSH ligand. Hydrodynamic experiments showed that GSH-AP column had less backpressure and plate height than those of GSH-FF column at high flow velocity, which was beneficial for its use in high-speed chromatography. The presence of flow-through pores in GSH-AP microspheres also accelerated the mass transfer rate of biomolecules induced by convective flow, leading to high protein resolution and high dynamic binding capacity (DBC) of glutathione S-transferase (GST) at high flow velocity. High purity of GST and GST-tagged recombinant human interleukin-1 receptor antagonist (rhIL-1RA) were obtained from crude extract with an acceptable recovery yield within 1.5min at a velocity up to 1400cm/h. GSH-AP medium is promising for high-speed affinity chromatography for the purification of GST and GST-tagged proteins.

Avian influenza H5 hemagglutinin binds with high avidity to sialic acid on different O-linked core structures on mucin-type fusion proteins

The interaction between P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG(2b)) fusion protein carrying multiple copies of the influenza hemagglutinin receptor Siaα2-3Gal on different O-glycan chains and recombinant human influenza H5N1 A/Vietnam/1203/04 hemagglutinin was investigated with a Biacore biosensor. The fusion protein was produced by stable cell lines in large scale cultures and purified with affinity- and gel filtration chromatography. TheC-P55 and 293-P cell lines were established by transfecting the Chinese hamster ovary (CHO)-K1 and Human embryonic kidney (HEK)-293 cell lines with plasmids encoding the PSGL-1/mIgG(2b) fusion protein, while the C-PSLex cell line was engineered by transfecting CHO-K1 cells with the plasmids encoding the core 2 β1,6GnT-I and FUT-VII glycosyltransferases. Glycosylation was characterized by lectin Western blotting of the proteins and liquid chromatography - mass spectrometry of released non-derivatized O-glycans. Biacore experiments revealed that PSGL-1/mIgG(2b) is a good binding partner of H5. The binding curves displayed a slow dissociation indicating a multivalent binding. The H5 hemagglutinin binds with similar strength to PSGL-1/mIgG(2b) carrying mostly sialylated core 1 (clone C-P55), a mix of sialylated core 1 and sialylated lactosamine (clone 293-P) or mainly sialylated lactosamine (clone C-PSLex) O-glycans, indicating that this hemagglutinin is unable to discriminate between these structures.The potential use of the large, flexible PSGL-1/mIgG(2b) mucin-type fusion protein carrying Siaα2-3Gal as a multivalent inhibitor of influenza virus is discussed.

In vivo intravascular biotinylation of Schistosoma bovis adult worms and proteomic analysis of tegumental surface proteins

Schistosoma bovis is a blood-dwelling fluke of ruminants that lives for years inside the vasculature of their hosts. The parasite tegument covers the surface of the worms and plays a key role in the host-parasite relationship. The parasite molecules expressed at the tegument surface are potential targets for immune or drug intervention. The purpose of this work was the identification of the proteins expressed in vivo on the surface of the tegument of S. bovis adult worms. To accomplish this we used a method based on in vivo vascular perfusion of mice infected with S. bovis which allowed the labelling of the surface of the worms inside the blood vasculature. The biotinylation of parasite inside blood vessels prevents the handling of worms in vitro and hence possible damage to the tegument that could produce results that would be difficult to interpret. Trypsin digestion of biotinylated proteins and subsequent liquid chromatography and tandem mass spectrometry analysis (LC-MS/MS) resulted in the identification on the S. bovis tegument of 80 parasite proteins and 28 host proteins. The proteins identified were compared with the findings from other proteomic studies of the schistosome surface. The experimental approach used in this work is a reliable method for selective investigation of the surface of the worms and provides valuable information about the exposed protein repertoire of the tegument of S. bovis in the environmental conditions that the parasite faces inside the blood vessels. To identify the proteins expressed on the surface of the tegument of S. bovis adult worms we used a method based on in vivo vascular perfusion, with biotin, of mice infected with S. bovis which allowed the labelling of the surface of the worms inside the blood vasculature. This methodology prevents the handling of worms in vitro and hence possible damage to the tegument that could produce results that would be difficult to interpret. This work is the first in which vascular perfusion has been used to investigate, in vivo, the protein exposed by an intravascular pathogen on its surface to the host, and provides valuable information about the exposed protein repertoire of the tegument of S. bovis in the environmental conditions that the parasite faces inside the blood vessels.

Characteristics and Application of Porous Ceramic/Agarose Composite Beads Derived as an Affinity Medium

Porous ceramic/agarose composite beads were derived as a kind of glutathione S-transferase (GST) affinity medium to investigate the characteristics and application in fast protein liquid chromatography. The analysis of back pressure and chromatographic performance in a packed bed indicated that this kind of affinity medium with a rigid structure and a high level of column efficiency would be suitable for protein chromatography under high flow velocity. The good physical stability evidenced under harsh alkaline treatments ensured the application in real chromatography processes. When the porous ceramic/agarose composite beads were used in the purification process of fusion protein GST-ADAM15, the purity of the total GST related protein reached more than 91.6 % and the yield reached 44.6 % even at the flow velocity of 764.3 cm h−1. The works indicated the characteristics of porous ceramic/agarose composite beads and their potential application in protein purification processes.

Post-mortem vitreous humour as potential specimen for detection of insulin analogues by LC–MS/MS

Differentiation of insulin analogues is required in forensic and clinical toxicology as well as in sports doping control. Immunoassay results provide only weak evidence for exogenous administration of insulin, as concentrations cannot be reliably interpreted and specific information on the insulin species remains unknown. In post-mortem blood, insulin degrades rapidly. In this study, improved methodology consisting of precipitation of proteins, immunoaffinity purification and liquid chromatography coupled to high resolution/high accuracy mass spectrometry were applied to post-mortem vitreous humour. Ten successive cases with a post-mortem interval from four to ten days were investigated for insulin analogues. The cause of death in these cases was connected with diabetes and its complications, as well as with chronic cardiovascular disease, alcoholism and cancer. In all cases, the manner of death was natural (disease). Insulin was positively detected in post-mortem vitreous humour in three cases out of ten by mass spectrometry. In two cases, the method revealed the long-acting insulin glargine (Lantus) metabolite M2 (DesB31-32 Lantus), and human insulin was detected in one case. The findings were in agreement with the documented history of insulin medication. No other obvious reason could be found for the failure of detecting insulins in the other cases than insulin degradation during the lengthy post-mortem interval. Vitreous humour is still a most prospective specimen for detection of insulin analogues post-mortem.

Purification of Coumarin Compounds From Cortex fraxinus by Adsorption Chromatography

In this paper, a chromatographic method for isolation and purification of coumarin compounds from Cortex fraxinus was established by using Superose 12 as the separation media for the first time. The conditions for separation were optimized. Four kinds of coumarin compounds including aesuletin, aesculin, fraxetin and fraxin were obtained. The purity of these compounds were 98.5, 99.1, 97.9 and 97.3%, respectively, which were determined by HPLC area normalization method. The chemical structures of the separated compounds were identified according to (1)H and (13)C nuclear magnetic resonance data. The retention behavior of the separated coumarin compounds on Superose 12 was also discussed. The retention is based on a mixture of hydrogen bonding and hydrophobic interactions between the coumarin compounds and the residues of the cross-linking reagents used in the manufacturing process of Superose 12. The results of this paper indicate that Superose 12 is not only suitable for size-exclusion chromatography of proteins and other biological macromolecules but also for low-molecular-weight natural products.

Preparation of porous styrenics-based monolithic layers for thin layer chromatography coupled with matrix-assisted laser-desorption/ionization time-of-flight mass spectrometric detection

Monolithic 50μm thin poly(4-methylstyrene-co-chloromethylstyrene-co-divinylbenzene) layers attached to 6.0cm×3.3cm glass plates have been prepared, using a thermally initiated polymerization process. These layers had a well-defined porous structure with a globular morphology demonstrated with SEM images and exhibited superhydrophobic properties characterized with a water contact angle of 157°. They were then used for thin-layer chromatography of peptides and proteins fluorescently labeled with fluorescamine. The spots of individual separated compounds were visualized using UV light, and their identities were confirmed with a matrix-assisted laser desorption/ionization time of flight mass spectrometry. The presence of chloromethylstyrene units in the polymer enabled hypercrosslinking via a Friedel–Crafts alkylation reaction, and led to monoliths with much larger surface areas, which were suitable for separations of small dye molecules.

RGS21, a regulator of taste and mucociliary clearance?

Motile cilia of airway epithelial cells help to expel harmful inhaled material. Activation of bitterant-responsive G protein-coupled receptors (GPCRs) is believed to potentiate cilia beat frequency and mucociliary clearance. In this study, we investigated whether regulator of G protein signaling-21 (RGS21) has the potential to modulate signaling pathways connected to airway mucociliary clearance, given that RGS proteins modulate GPCR signaling by acting as GTPase-accelerating proteins (GAPs) for the Gα subunits of heterotrimeric G proteins. This is a pilot investigation to determine if RGS21, a potential tastant specific RGS gene, is expressed in sinonasal mucosa, and to determine its specific Gα substrate using in vitro biochemical assays with purified proteins. Rgs21 expression in sinonasal mucosa was determined using quantitative, real-time PCR and a transgenic mouse expressing RFP from the Rgs21 promoter. Rgs21 was cloned, over-expressed, and purified using multistep protein chromatography. Biochemical and biophysical assays were used to determine if RGS21 could bind and accelerate the hydrolysis of GTP on heterotrimeric Gα subunits. Rgs21 was expressed in sinonasal mucosa and lingual epithelium. Purified recombinant protein directly bound and accelerated GTP hydrolysis on Gα subunits. Rgs21 is expressed in sinonasal mucosa, is amenable to purification as a recombinant protein, and can bind to Gα(i/o/q) subunits. Furthermore, RGS21 can accelerate the hydrolysis rate of GTP on Gαi subunits. This provides evidence that RGS21 may be a negative regulator of bitterant responses. Future studies will be needed to determine the physiological role of this protein in mucociliary clearance.

Monolithic cryopolymers with embedded nanoparticles. II. Capillary liquid chromatography of proteins using charged embedded nanoparticles

The preparation of composite monolithic cryopolymers is presented. These novel porous materials were prepared in capillary format at −70°C using poly(ethyleneglycol) diacrylate (PEGDA) Mw 258 as the single monomer and a mixture of dioxane and water as the porogen. Positively (NR4+) or negatively (SO3−) charged nanoparticles were incorporated within the polymeric structure by direct addition of their suspensions to the polymerisation mixture. In contrast to our previous report using neutral nanoparticles, the trapping of charged nanoparticles is mostly observed at the polymer surface. The incorporation of these nanostructures improved the chromatographic separations of standard proteins under a hydrophobic interaction chromatography (HIC) separation mode. Moreover, the presence of ionic groups on the polymer surface allowed the application of these columns under ion-exchange (IEX) conditions. The results obtained in this work show that the functionalisation of monolithic columns by direct addition of nanoparticles is a good alternative towards the modification of monolithic polymers without altering the polymeric scaffold.

High‐throughput process development methods for chromatography and precipitation of proteins: Advantages and precautions

Although high‐throughput process development methods using 96‐well microplate (MP) systems are promising and attractive, they still have several unclear problems in the practical applications. Several case studies for the purification process development by using manually operated 96‐well MP systems are presented. In the first example, the MPs with ion‐exchange monolith disks or ion‐exchange membranes were employed for linear gradient elution and breakthrough curve experiments. The data such as the peak salt concentration and dynamic binding capacity values obtained with the MP system were in good agreement with those by the LC system. In the second example, batch adsorption experiments with a 96‐well filter MP were performed for a protein A resin (particle)–antibody system in order to obtain the adsorption isotherms. The static binding capacities calculated from the isotherms were similar to those calculated from the LC system. In the last example, protein precipitation experiments by PEG were carried out for determining the solubility curve. It was found that the presence of dimer and aggregates affect the solubility curve measurement significantly. The proposed methods suggest powerful practical applications of high‐throughput process development for chromatography and precipitation process when the experiments are carefully designed and carried out.