Thiophilic Adsorption Chromatography Research Articles

Purification of Anti-Mycobacterium tuberculosis MPT64 Immunoglobulin-Y from Egg-Yolk Supernatant Using Thiophilic Adsorption Chromatography

The significance of immunoglobulin Y (IgY) as a particular antibody equal to mammalian IgG is well understood. However, due to a lack of reliable purification procedures, producing highly pure IgY remains problematic. In this study, we aimed to optimize the recovery of pure IgY anti-MPT64 using thiophilic adsorption chromatography. The purification of IgY anti-MPT64 was achieved by initial PEG lipid precipitation, then an optimized purification by varying the gradient concentration of elution buffer into five steps gradient (0-20, 20-40, 40-60, 60-80, and 80-100%v/v) for three injection column volume (CV) each and two steps gradient (0-50 and 50-100%v/v) in eight CV for each concentration. The obtained IgY was characterized by SDS-PAGE and dot-blot then determined its content levels using the Lowry method. The results showed that the five steps gradient purification was found to provide a better purity level of IgY than the two steps gradient. However, the IgY content obtained in the two steps gradient purification (2.2632± 0.011 mg/mL) was higher than the five steps gradient purification (1.35482 ± 0.023 mg/mL). Nevertheless, both purified IgY results can recognize MPT64 protein through a dot blot test. Therefore, it can be summarized that thiophilic adsorption chromatography with five steps gradient of purification was an efficient process to obtain a higher purity of IgY anti-MPT64, especially to be targeted as a diagnostic kit component for MPT64 detection.

Development of SarS-Cov-2 antigen Detection Kit Based on Immunoglobulin Y (Igy) Using Surface Plasmon resonance (SPr)

SARS disease reappeared at the end of 2019 with a new name as Coronavirus Disease 2019 (COVID-19) caused by a new virus called SARS-CoV-2. This virus has spread throughout the world until recently and caused massive deaths and losses. The nucleic acid test in the form of real-time reverse transcriptase-polymerase chain reaction (RT-PCR) is very important to diagnose COVID-19 in patients, but this method has several drawbacks such as operators who have to be trained, the diagnosis results appear in a relatively long time, and the examination price relatively expensive. This research was conducted to produce immunoglobulin Y (IgY) extracted from chicken egg yolk targeting the S-protein receptor-binding domain (RBD) on SARS-CoV-2 as a component of the surface plasmon resonance (SPR) SARS-CoV-2 antigen detection kit. This research was started by extracting IgY from hyperimmune chicken egg yolks with the polyethylene glycol (PEG) precipitation method and continued with dialysis. The extracted IgY was further purified using thiophilic adsorption chromatography and concentrated by using Amicon® Ultra-15 ultrafiltration. The IgY activity against SARS-CoV-2 RBD was tested qualitatively using the agar gel precipitation test (AGPT) technique and the total protein content was determined using the Lowry method. IgY was tested for its affinity against SARS-CoV-2 RBD using SPR. The IgY concentration obtained was 11 mg/mL. The AGPT result showed the presence of IgY activity against SARS-CoV-2 RBD isolated from egg yolk and chicken serum after 8 weeks after the first vaccination of chickens. The SDS-PAGE results showed a very clear band of IgY characters. The obtained IgY showed adequate interaction with commercial SARS-CoV-2 RBD on an SPR device. The purified IgY was able to bind with protein-S RBD and showed a fairly good affinity for the SARS-CoV-2 antigen sample. The results of these observations indicate that IgY anti-S-protein SARS-CoV-2 can be produced and purified from chicken egg yolk and can be used as a diagnostic component to detect SARS-CoV-2 antigen, especially on SPR.

Purification of Native and Recombinant Cobra Venom Factor Using Thiophilic Adsorption Chromatography

The complement activating venom component Cobra Venom Factor (CVF) forms a stable CVF-dependent C3 convertase complex, which initiates continuous activation of the complement system, consumes all downstream complement components and obliterates functional complement. Therefore, native CVF is routinely used as decomplementing agent in vivo and in vitro. However, in most countries, CVF and even unfractionated cobra venom are now becoming unavailable due to the CITES agreement. Although CVF is a complex molecule with three disulfide linked polypeptide chains and pronounced glycosylation, recombinant expression of the active molecule in eukaryotic host cells may provide an alternative source. In this study we describe a strategy for the production and efficient isolation of recombinant CVF from supernatant of mammalian cells. Thiophilic adsorption chromatography (TAC), an efficient procedure for purification of the human homologue C3, was evaluated for its suitability regarding purification of both native as well as recombinant CVF. Native CVF could be purified by TAC in a one-step procedure from cobra venom with yields of 92% compared to 35% by conventional approaches. After establishment of stably transfected mammalian cells recombinant CVF could be obtained and enriched from CHO supernatants by TAC to a purity of 73%, and up to 90% if an additional affinity chromatography step was included. Subsequent characterization revealed comparable hemolytic and bystander lysis activity and of rCVF and nCVF. These data demonstrate that the functional expression in mammalian cells in combination with TAC for purification renders rCVF a highly attractive substitute for its native counterpart.

Chromatographic and fluorometric study of interactions between thiophilic and hydrophobic ligands and tryptophan peptide homologues

The interactions of tryptophan and its peptide homologues with thiophilic ligands were studied in terms of their chromatographic retention and steady-state fluorescence under various conditions, and compared with non-polar structures typically regarded as pure hydrophobic ligands. The experimental results show that both non-polar and polar interactions are involved in what has been termed “thiophilic adsorption chromatography”.

Serpins of oat (Avena sativa) grain with distinct reactive centres and inhibitory specificity.

Most proteinase inhibitors from plant seeds are assumed to contribute to broad-spectrum protection against pests and pathogens. In oat (Avena sativa L.) grain the main serine proteinase inhibitors were found to be serpins, which utilize a unique mechanism of irreversible inhibition. Four distinct inhibitors of the serpin superfamily were detected by native PAGE as major seed albumins and purified by thiophilic adsorption and anion exchange chromatography. The four serpins OSZa-d are the first proteinase inhibitors characterized from this cereal. An amino acid sequence close to the blocked N-terminus, a reactive centre loop sequence, and the second order association rate constant (ka') for irreversible complex formation with pancreas serine proteinases at 24 degrees C were determined for each inhibitor. OSZa and OSZb, both with the reactive centre scissile bond P1-P1' Thr downward arrow Ser, were efficient inhibitors of pancreas elastase (ka' > 105M-1 s-1). Only OSZb was also an inhibitor of chymotrypsin at the same site (ka' = 0.9 x 105M-1 s-1). OSZc was a fast inhibitor of trypsin at P1-P1' Arg downward arrow Ser (ka' = 4 x 106M-1 s-1); however, the OSZc-trypsin complex was short-lived with a first order dissociation rate constant kd = 1.4 x 10-4 s-1. OSZc was also an inhibitor of chymotrypsin (ka' > 106M-1 s-1), presumably at the overlapping site P2-P1 Ala downward arrow Arg, but > 90% of the serpin was cleaved as substrate. OSZd was cleaved by chymotrypsin at the putative reactive centre bond P1-P1' Tyr downward arrow Ser, and no inhibition was detected. Together the oat grain serpins have a broader inhibitory specificity against digestive serine proteinases than represented by the major serpins of wheat, rye or barley grain. Presumably the serpins compensate for the low content of reversible inhibitors of serine proteinases in oats in protection of the grain against pests or pathogens.

Use of thiophilic adsorption chromatography for the one-step purification of a bacterially produced antibody F(ab) fragment without the need for an affinity tag.

Thiophilic adsorption chromatography (TAC) was employed for the purification of a recombinant F(ab) fragment of the antibody IN-1 from the periplasmic protein fraction of Escherichia coli. Adsorption of the F(ab) fragment to the T-gel was achieved at a high concentration of ammonium sulfate and turned out to be independent of the presence of a His(6) tag or Strep tag or of the human or murine nature of the C(H)1 and C(L) domains (subclass IgG1/kappa). Elution was effected by means of a decreasing salt gradient, yielding fractions with the correctly assembled, heterodimeric F(ab) fragment at high purity. Interestingly, the single substitution of an alanine residue with phenylalanine in the CDR-L1 of the F(ab) fragment significantly enhanced the retention on the column so that quantitative elution necessitated prolonged application of a low-salt buffer. Our findings suggest that TAC is generally suitable for the isolation of bacterially produced F(ab) fragments and support the notion that aromatic side chains play an important role in the interaction with the affinity matrix. This method should prove valuable in the production of proteins for in vivo applications as might be the case for the F(ab) fragment of the antibody IN-1, which promotes axonal regeneration in the central nervous system.

Papers to Appear in Forthcoming Issues

Expression and Purification of the BmK M1 Neurotoxin from the Scorpion Buthus martensii KarschFeng Shao, Yu-Mei Xiong, Rong-Huan Zhu, Min-Hua Ling, Cheng-Wu Chi, and Da-Cheng WangMolecular Cloning of the Arylsulfate Sulfotransferase Gene and Characterization of Its Product from Enterobacter amnigenus AR-37Ae-Ran Kwon, Tae-Gwon Oh, Dong-Hyun Kim, and Eung-Chil ChoiExpression and Purification of the Transcription Factor NtcA from the Cyanobacterium Anabaena PCC 7120Susanne Wisén, Fanyi Jiang, Birgitta Bergman, and Bengt MannervikIn Vitro Refolding of Heterodimeric CapZ Expressed in E. coli as Inclusion Body ProteinKirsten Remmert, Detlef Vullhorst, and Horst HinssenUse of Carboxypeptidase Y Propeptide as a Fusion Partner for Expression of Small Polypeptides in Escherichia coliGui Hwan Oh, Moon Sun Hahm, and Bong Hyun ChungExpression and Purification of the Hormone Binding Domain of the Drosophila Ecdysone and Ultraspiracle ReceptorsB. P. Halling, D. A. Yuhas, R. R. Eldridge, S. N. Gilbey, V. A. Deutsch, and J. D. HerronBacterial Expression and Characterization of Functional Recombinant Triosephosphate Isomerase from Schistosoma japonicumWenYu Sun, ShuXian Liu, Paul J. Brindley, and Donald P. McManusLarge-Scale HPLC Purification of Calbindin D9k from Porcine IntestineValentina Bonetto, Liina Kangur, Peep Palumaa, Viktor Mutt, Hans Jörnvall, and Rannar SillardUse of Thiophilic Adsorption Chromatography for the One-Step Purification of a Bacterially Produced Antibody Fab Fragment without the Need for an Affinity TagMarkus Fiedler and Arne Skerra

Performance of thiophilic adsorption chromatography in the purification of rat IgG2b monoclonal antibodies from serum- and protein-free culture supernatants.

The usefulness of thiophilic adsorption chromatography for the purification of rat IgG2b monoclonal antibodies has been evaluated. This approach has not shown specificity for immunoglobulins; therefore, to minimize potential interferences, the purification was carried out from supernatants of hybridomas grown in serum- and protein-free conditions. The protein purity of the six final antibody preparations assayed was always >/= 97%. In addition to the easiness of this procedure, which enables one-step antibody purification, the materials employed are rather inexpensive and milligram amounts of antibody can be recovered from 1 L of supernatant. Overall, the purification of rat IgG2b monoclonals under the conditions reported here offers an advantageous alternative to other more expensive and cumbersome methods.

Performance of thiophilic adsorption chromatography in the purification of rat IgG2b monoclonal antibodies from serum‐ and protein‐free culture supernatants

The usefulness of thiophilic adsorption chromatography for the purification of rat IgG2b monoclonal antibodies has been evaluated. This approach has not shown specificity for immunoglobulins; therefore, to minimize potential interferences, the purification was carried out from supernatants of hybridomas grown in serum- and protein-free conditions. The protein purity of the six final antibody preparations assayed was always ≥97%. In addition to the easiness of this procedure, which enables one-step antibody purification, the materials employed are rather inexpensive and milligram amounts of antibody can be recovered from 1 L of supernatant. Overall, the purification of rat IgG2b monoclonals under the conditions reported here offers an advantageous alternative to other more expensive and cumbersome methods. Copyright © 1999 John Wiley & Sons, Ltd.

Thiophilic adsorption chromatography: purification of Equ c2 and Equ c3, two horse allergens from horse sweat

Purification of two allergens from horse ( Equus caballus) sweat, Equ c2 and Equ c3, by means of salt-promoted chromatography on a “thiophilic” (T-gel) adsorbent is described. Immobilization of these proteins was found to be dependent on the presence of water-structure-forming salts where the ammonium sulphate concentration in the equilibration buffer was 2 M. Equ c2 showed higher affinity towards the thiophilic matrix than Equ c3. Their molecular mass ( M r) values established by SDS–polyacrylamide gel electrophoresis were for Equ c2 ≈17 000 and for Equ c3 ≈16 000, and both proteins showed a low isoelectric point of ≈3.8. Their allergenic properties were also investigated using sera from horse-sensitized patients, where it was demonstrated that these proteins exhibited an IgE antibody binding capacity. In this report we show the broad potential applications of thiophilic adsorption chromatography for the efficient purification of allergens.

Presence of antibodies specific for proteins ofContracaecum osculatum(Rudolphi, 1908) in plasma of several Antarctic teleosts

The immunoglobulin fraction was purified from the plasma of an Antarctic teleost,Chionodraco hamatus(Lönnberg, 1906) by thiophilic adsorption chromatography and size-exclusion FPLC. Somatic proteins ofContracaecum osculatumlarvae were recovered by boiling whole nematodes in phosphate buffer containing 2% SDS and 10% β-mercaptoethanol. Immunoglobulins fromC. hamatusplasma recognisedC. osculatumsomatic antigens either blotted onto nitrocellulose or immobilised on a HiTrap NHS-activated column. The relative molecular mass of the majorC. osculatumantigen was 91kDa. Plasma from specimens ofC. hamatus,Notothenia coriiceps(Richardson, 1844),Notothenia gibberifrons(Lönnberg, 1905),Chaenocephalus aceratus(Lönnberg, 1906),Pseudochaenichthys georgianus(Norman, 1937),Trematomus bernacchii(Boulenger, 1902) were tested in double immunodiffusion againstC. osculatumproteins. Immunoprecipitates were observed with individual plasma of five out of six species. The results demonstrate the presence of nematode-specific antibodies in Antarctic fish plasma.

Comparison of the protein adsorption selectivity of salt-promoted agarose-based adsorbents: Hydrophobic, thiophilic and electron donor–acceptor adsorbents

Protein adsorption of human serum onto six different agarose-based chromatographic gels that were representative of the salt-promoted adsorbent family [octyl- and phenyl-Sepharose, mercaptoethanol–divinyl sulfone agarose (T gel), mercaptomethylene pyridine-derivatized agarose gel (MP gel), tricyanoaminopropene–divinyl sulfone agarose (DVS–TCP gel), tricyanoamino-propene–bisoxirane agarose (bisoxirane–TCP gel)] was studied in the presence of moderate or high concentrations of the water structuring salt, sodium sulfate. Study of the protein adsorption selectivity by two-dimensional gel electrophoresis revealed an opposed selectivity for hydrophobic interaction adsorbents and electron donor–acceptor adsorbents. The T gel, MP gel and TCP gels belonged to the electron donor–acceptor adsorbents, displaying a main selectivity for immunoglobulins, whereas octyl-Sepharose belonged to the hydrophobic adsorbents, displaying a main selectivity for `hydrophobic' proteins. Phenyl-Sepharose for its part was described as an example of a composite selectivity of both families. The conclusion of this work is two-fold: (1) hydrophobic interaction chromatography (HIC) and electron donor–acceptor chromatography (EDAC) have opposed protein selectivities and are both salt-promoted. As a main consequence, it means that high concentrations of a water-structuring salt can promote different types of weak molecular interactions, resulting in different protein adsorption selectivities: (2) thiophilic adsorption chromatography (TAC) should be renamed EDAC as similar protein selectivity is demonstrated for electron donor–acceptor ligand devoid of sulfur atoms.

Five-membered mercaptoheterocyclic ligands for thiophilic adsorption chromatography.

A series of five-membered mercaptoheterocyles containing at least two heteroatoms in their ring structure was investigated for their ability to specifically adsorb antibodies. The structures that contain at least one double bond adsorbed antibodies, while the saturated structure showed no protein interaction under the experimental conditions studied. The influence of various lyotropic salt on adsorbance was studied.

Isolation of Human Serum IgA Using Thiophilic Adsorption Chromatography

Isolation of Human Serum IgA Using Thiophilic Adsorption Chromatography

Immunoglobulin and non-immunoglobulin components of human milk inhibit Clostridium difficile toxin A-receptor binding.

Clostridium difficile is isolated from the intestinal tracts of > 50% of healthy infants. The mechanism by which intestinal colonisation of infants by toxigenic C. difficile is generally asymptomatic is unknown but may reflect the presence in human milk of neutralising activity against C. difficile toxin A. On this basis, the ability of human milk to inhibit the binding of toxin A to a purified hamster brush border membrane receptor was determined. Ten milk samples from healthy volunteers in various stages of lactation inhibited the binding of toxin A to the receptor by an average of 90%. Heating and dialysis did not significantly alter the inhibitory activity of any of the milk samples. Human milk protected adult hamsters against a lethal challenge with toxin A but had no effect on the cytotoxic activity of the toxin. SDS-PAGE and ligand blot analyses showed that there were at least four distinct factors in human milk that specifically bound toxin A. Thiophilic adsorption chromatography was used to separate immunoglobulin from non-immunoglobulin components of human milk. IgA was the only immunoglobulin detected in human milk and > 90% of this immunoglobulin was recovered after purification by thiophilic adsorption. Both the unbound non-immunoglobulin and bound immunoglobulin fractions of human milk inhibited the binding of toxin A to the purified receptor. These results suggest that human milk may be important in protecting infants against C. difficile-associated intestinal disease.

Purification of human procollagen type I carboxyl-terminal propeptide cleaved as in vivo from procollagen and used to calibrate a radioimmunoassay of the propeptide

We purified human procollagen type I carboxyl-terminal propeptide (PICP) that had been cleaved as in vivo from procollagen. PICP in serum-free medium from cultured human fetal fibroblasts was purified by thiophilic adsorption chromatography, low-pressure gel filtration, and HPLC gel filtration. The purity and homogeneity of the protein was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal amino acid sequencing showed that the sequences of the alpha 1 and alpha 2 chains of this PICP were identical to those of the PICP produced in vivo. The monocomponent PICP thus purified was used as calibrator in a simple equilibrium-type RIA of PICP with polyclonal antibodies raised in rabbits. The measuring range is 0.15-3.75 nmol/L, and the assay detection limit is 0.03 nmol/L. The within-run and total CVs are 2% and 4%, respectively. The reference interval for the plasma concentration of PICP in healthy women of ages > 30 years is 0.36-1.44 nmol/L (geometric mean 0.72 nmol/L, n = 154).

Serpins from wheat grain

Wheat serpin genes have been identified by Southern blot hybridization with three distinct barley protein Z probes. Immunoblot analysis with a monoclonal antibody towards barley protein Z confirmed expression of related M r ~ 40 kDa proteins in wheat grain. The wheat serpins were extracted under reducing conditions and separated from β-amylase and other seed proteins by thiophilic adsorption and anion-exchange chromatography. One molecular form possessing chymotrypsin inhibitory activity was isolated in a reactive site cleaved form on a chymotrypsin affinity column. N-terminal amino acid sequences of a CNBr fragment and of the C-terminal peptide from the cleaved inhibitor (M r 4574 ± 4 Da) verified homology with barley protein Z and mammalian serpins. The native inhibitory serpin was demonstrated to form an SDS-stable complex with α-chymotrypsin.

Sulfone-aromatic ligands for thiophilic adsorption chromatography: Purification of human and mouse immunoglobulins

New thiophilic matrices and new procedures were used for the purification of immunoglobulins both from human serum and from hybridoma cell cultures containing fetal calf serum. A range of aromatic and heteroaromatic ligands containing hydroxyl or amino groups have been coupled to divinyl sulfone-activated agarose. The resulting affinity matrices have the general formula M-OCH 2CH 2SO 2CH 2CH 2- X- Y, where M is the agarose matrix, X is oxygen or nitrogen, and Y is an aromatic or heteroaromatic compound. Contrary to earlier expectations these matrices showed pronounced thiophilic binding patterns when tested for the selective binding of immunoglobulins from human serum. The binding is influenced by the structure of the aromatic part of the ligand, the ligand concentration, and the concentration and type of lyotropic salt. 2-Hydroxypyridine coupled to divinyl sulfone-activated agarose was used to purify murine monoclonal antibodies (IgG 1 and IgM) from hybridoma cell cultures containing fetal calf serum. Compared to previous methods, significantly increased binding capacity (300–1500%) was obtained by using 1.0–1.2 m ammonium sulfate. Purity of the monoclonal antibody may be optimized for each individual clone by washing the column with either a low concentration of ammonium sulfate or polyethylene glycol before elution.

Application of phase partitioning and thiophilic adsorption chromatography to the purification of monoclonal antibodies from cell culture fluid

A two-step method for the isolation of an IgG1 monoclonal antibody against horseradish peroxidase from hybridoma cell culture supernatant is described. Purification was achieved using an aqueous two-phase extraction procedure in conjunction with thiophilic adsorption chromatography. In an aqueous two-phase system composed of 5% PEG 1540 and 22% phosphate the monoclonal antibody preferentially associates with the PEG-rich top phase whereas proteins such as albumin and transferrin partition into the salt-rich bottom phase. Final purification of the monoclonal antibody was achieved by subjecting the PEG-rich top phase to thiophilic adsorption chromatography. The monoclonal antibody purified to homogeneity retained its specificity for horseradish peroxidase as revealed by polyacrylamide gel electrophoresis and an enzyme-linked immunosorbent assay. The potential of this purification protocol for large scale applications is discussed.

Thiophilic adsorption chromatography: The separation of serum proteins

Human serum proteins were separated on a matrix obtained by reaction of β-mercaptoethanol with divinyl sulfone-activated agarose (the so-called T-gel). Binding of almost all serum proteins was observed at high concentrations of ammonium sulfate. Elution was achieved by gradually lowering the concentration of salt in the washing buffer. Fractions obtained during elution were analyzed by fused rocket immunoelectrophoresis. Proteins were recovered in high yields and with an excellent separation in this one-step procedure (“Thiophilic adsorption chromatography”). A rapid and straightforward procedure giving essentially pure immunoglobulins from crude rabbit serum with at least 80% yield by the T-gel is also presented.