Affinity Chromatography Matrix Research Articles

Purification and Biochemical Characterization of Novel Galectin from the Black Poplar Medicinal Mushroom Cyclocybe cylindracea (Agaricomycetes) Strain MEST42 from Algeria.

In the present study, a new galectin designated Cyclocybe cylindracea lectin (CCL) was extracted from the fruiting bodies of the wild black popular mushroom C. cylindracea grown in Algeria. The protein was isolated using sepharose 4B as affinity chromatography matrix, and galactose as elutant. The purified galectin was composed of two subunits of 17.873 kDa each, with a total molecular mass of 35.6 kDa. Its agglutinant activity was impeded by galactose and its derivatives, as well as melibiose. Lactose showed the highest affinity, with a minimal inhibitory concentration of 0.0781 mM. CCL was sensitive to extreme pH conditions, and its binding function decreased when incubated with 10 mM EDTA, and it could be restored by metallic cations such as Ca2+, Mg2+, and Zn2+. CCL agglutinated human red blood cells, without any discernible specificity. Circular dichroism spectra demonstrated that its secondary structure contained β-sheet as dominant fold. In addition, bioinformatics investigation on their peptide fingerprint obtained after MALDI-TOF/TOF ionization using mascot software confirmed that CCL was not like any previous purified lectin from mushroom: instead, it possessed an amino acid composition with high similarity to that of the putative urea carboxylase of Emericella nidulans (strain FGSC A4/ATCC 38163/CBS 112.46/NRRL 194/M139) with 44% of similarity score.

A high-throughput screening strategy identifies adenosine 5'-monophosphate as a novel enhancing molecule of the cardio-protective enzyme 5-oxoprolinase (OPLAH)

Abstract Background/Introduction Oxidative stress plays an essential role in the pathophysiology of heart failure (HF), and reducing it might improve outcomes in patients suffering from it. We recently identified 5-oxoproline to be elevated in patients with HF and that the increased levels are associated with a poor prognosis. 5-oxoprolinase (OPLAH) is an enzyme that converts toxic 5-oxoproline to harmless glutamate. We previously showed that the overexpression of OPLAH prevented cardiac damage in experimental models. Purpose To identify small molecules that hold the potential to increase the activity of the antioxidant enzyme OPLAH. Methods and results OPLAH expression vectors were established containing a 6xHis-tag in the N-terminal region and were used to efficiently overexpress enzymatically active recombinant OPLAH protein (rOPLAH) in human embryonic kidney cells (HEK293A) and Spodoptera frugiperda cells (SF9). rOPLAH expressing His-tag was purified under native conditions using an affinity chromatography matrix, and enzymatic activity was proven by measuring the conversion of 5-oxoproline to glutamate using LC-MS/MS. After miniaturization and optimization of a luminescence-based technique that measures OPLAH's ATPase activity, a high-throughput screening campaign of 1280 FDA-approved compounds was performed. Adenosine 5′-monophosphate (5'-AMP) was identified to enhance the ATPase activity of OPLAH by >50%. In addition, an LC-MS/MS dose-response curve exhibited that increasing 5'-AMP concentration resulted in a higher glutamate production using isolated rOPLAH. A thermal shift assay confirmed the interaction of 5'-AMP and OPLAH. Since 5’-AMP is unable to cross the cellular plasma membrane, acetalated dextran spermine-modified nanoparticles loaded with 5'-AMP were produced (AcDXSp@AMP NPs) to target cells. NPs were prepared by the water-in-oil-in-water double emulsion technique. Particle size, polydispersity index and z–potential of the NPs were characterized by dynamic and electrophoretic light scattering. HPLC tested the quantification of encapsulated 5'-AMP and its release over time. The efficacy of OPLAH activation by 5'-AMP-loaded NPs was tested in HeLa cells cultured in the presence of 10 mM 13C-5-oxoproline. Employing AcDXSp@AMP NPs for 12h to cells resulted in about 50% elevation of 13C-glutamate production, showing successful activation of OPLAH by AMP in cells. Conclusion Here, we developed a high-throughput screening for 5-oxprolinase and identified a non-cell permeable enhancer compound, adenosine 5′-monophosphate. Orthogonal studies confirmed 5'-AMP affinity to OPLAH. Finally, we successfully utilized nanoparticles as a drug-delivery system for 5'-AMP and proved its intracellular effect. Our efforts are a blueprint for bringing target discovery to treatment identification. As such, nanoparticle-based delivery systems hold great potential for future trials utilizing impermeable modulators to target heart failure and other diseases.Illustrative workflow

Implementation of Novel Affinity Ligand for Lentiviral Vector Purification.

The use of viral vectors as therapeutic products for multiple applications such as vaccines, cancer treatment, or gene therapies, has been growing exponentially. Therefore, improved manufacturing processes are needed to cope with the high number of functional particles required for clinical trials and, eventually, commercialization. Affinity chromatography (AC) can be used to simplify purification processes and generate clinical-grade products with high titer and purity. However, one of the major challenges in the purification of Lentiviral vectors (LVs) using AC is to combine a highly specific ligand with a gentle elution condition assuring the preservation of vector biological activity. In this work, we report for the first time the implementation of an AC resin to specifically purify VSV-G pseudotyped LVs. After ligand screening, different critical process parameters were assessed and optimized. A dynamic capacity of 1 × 1011 total particles per mL of resin was determined and an average recovery yield of 45% was found for the small-scale purification process. The established AC robustness was confirmed by the performance of an intermediate scale providing an infectious particles yield of 54%, which demonstrates the scalability and reproducibility of the AC matrix. Overall, this work contributes to increasing downstream process efficiency by delivering a purification technology that enables high purity, scalability, and process intensification in a single step, contributing to time-to-market reduction.

Cofactor self-sufficient by co-immobilization of pyridoxal 5′-phosphate and lysine decarboxylase for cadaverine production

Cadaverine (1,5-diaminopentane) has widespread applications in industry, agriculture and medicine. Cadaverine can be biotransformed from l-lysine by lysine decarboxylasey which is a pyridoxal 5′-phosphate (PLP)-dependent enzyme. Herein, barium alginate microspheres were fabricated by electro-dripping and employed as immobilized metal ion affinity chromatography matrix to purify and immobilize engineered lysine decarboxylase CadA-EK. Besides, polyethyleneimine (PEI) was employed for co-immobilizing PLP and enzymes (cCadA-EK-PEI/PLP). The cCadA-EK-PEI/PLP can produced 184.53 g/L cadaverine with yield of 90.03% in 2 mol/L lysine solution. And the stability of co-immobilized CadA-EK was 1–4 times higher than that of iCadA-EK + PLP in environmental factors, and the catalytic efficiency kcat/Km was increased by 3.4-fold. Furthermore, a continuous flow microreactor with the co-immobilized enzymes and cofactors was developed to catalyze lysine, with 14.28 g/(L·h) productivity of cadaverine, which displayed great potential for cadaverine biosynthesis. This work provides alternative strategy for the one-step purification and co-immobilization of enzyme and cofactor.

Design of Affinity Chromatography Peptide Ligands Through Combinatorial Peptide Library Screening.

In this chapter, a protocol to design affinity chromatography matrices with short peptide ligands immobilized for protein purification is described. The first step consists of the synthesis of a combinatorial peptide library on the hydroxymethylbenzoyl (HMBA)-ChemMatrix resin by the divide-couple-recombine (DCR) method using the Fmoc chemistry. Next, the library is screened with the protein of interest labeled with a fluorescent dye or biotin. Subsequently, peptides contained on positive beads are identified by tandem matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS/MS), and those sequences showing greater consensus are synthesized in larger quantities and immobilized on chromatographic supports. Finally, target protein adsorption on peptide affinity matrices is evaluated through equilibrium adsorption isotherms and breakthrough curves.

Single-step purification of equine chorionic gonadotrophin directly from plasma using affinity chromatography

Equine chorionic gonadotrophin (eCG) is a hormone widely used in superovulation protocols because of its follicle-stimulating action, which increases reproductive efficiency in animals of productive interest. It contains 45% carbohydrate, 10% of which is N-acetylneuraminic acid (sialic acid). The eCG purification procedures from equine serum or plasma are mainly based on chromatographic methods. However, before these procedures, it is necessary to follow sample pre-conditioning steps, such as several precipitation stages and/or ultrafiltration/diafiltration processes. In this work, an efficient affinity chromatographic matrix for eCG purification directly from plasma was developed. The matrix consisted of chitosan mini-spheres with immobilized wheat germ agglutinin (WGA). The matrix allowed 98% adsorption of eCG directly from plasma without any pre-treatment with an overall yield of around 60%. The matrix chosen was able to maintain the efficient performance of the purification process for three consecutive cycles. Also, the process was scaled-up 500 times in volume and tested over seven consecutive cycles maintaining its chromatographic performance. The results presented here suggest the potential application of this matrix to one-step purification of eCG from plasma.

Immobilized metal affinity chromatography matrix modified by poly (ethylene glycol) methyl ether for purification of angiotensin I-converting enzyme inhibitory peptide from casein hydrolysate

Purification of small bioactive peptides from complex biological samples is a difficult task due to the interference of concentrated large biomolecules. In this study, a magnetic immobilized metal affinity chromatography matrix modified by poly (ethylene glycol) methyl ether (IMACM@mPEG) was prepared and applied for the rapid purification of angiotensin I-converting enzyme (ACE) inhibitory peptides from casein hydrolysate. The proposed IMACM@mPEG considerably reduced the non-specific adsorption of large proteins and exhibited improved purification efficiency towards ACE inhibitory peptides. A novel peptide with moderate ACE inhibitory activity (IC50 value of 274 ± 5 μM) was identified as LLYQEPVLGPVR. Lineweaver-Burk plot confirmed the non-competitive inhibition pattern of LLYQEPVLGPVR. The purified peptide was digested after simulated gastrointestinal digestion and produced shorter peptides which contributed to enhanced ACE inhibitory activity. These results indicated that the IMACM@mPEG is an effective method for the prepurification of ACE inhibitory peptide and the purified peptide LLYQEPVLGPVR may have potential as nutraceutical ingredient in functional foods for hypertension treatments.

Expression, purification and virucidal activity of two recombinant isoforms of phospholipase A2 from Crotalus durissus terrificus venom.

The global emergence and re-emergence of arthropod-borne viruses (arboviruses) over the past four decades have become a public health crisis of international concern, especially in tropical and subtropical countries. A limited number of vaccines against arboviruses are available for use in humans; therefore, there is an urgent need to develop antiviral compounds. Snake venoms are rich sources of bioactive compounds with potential for antiviral prospection. The major component of Crotalus durissus terrificus venom is a heterodimeric complex called crotoxin, which is constituted by an inactive peptide (crotapotin) and a phospholipase A2 (PLA2-CB). We showed previously the antiviral effect of PLA2-CB against dengue virus, yellow fever virus and other enveloped viruses. The aims of this study were to express two PLA2-CB isoforms in a prokaryotic system and to evaluate their virucidal effects. The sequences encoding the PLA2-CB isoforms were optimized and cloned into a plasmid vector (pG21a) for recombinant protein expression. The recombinant proteins were expressed in the E. coli BL21(DE3) strain as insoluble inclusion bodies; therefore, the purification was performed under denaturing conditions, using urea for protein solubilization. The solubilized proteins were applied to a nickel affinity chromatography matrix for binding. The immobilized recombinant proteins were subjected to an innovative protein refolding step, which consisted of the application of a decreasing linear gradient of urea and dithiothreitol (DTT) concentrations in combination with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS) as a protein stabilizer. The refolded recombinant proteins showed phospholipase activity and virucidal effects against chikungunya virus, dengue virus, yellow fever virus and Zika virus.

The effects of Enterolobium contortisiliquum serine protease inhibitor on the survival of the termite Nasutitermes corniger, and its use as affinity adsorbent to purify termite proteases.

The immobilization of Enterolobium contortisiliquum protease inhibitor, EcTI-Sepharose, as an affinity chromatography matrix is a powerful biotechnological tool to purify targets from Nasutitermes corniger in the investigation of insecticidal properties of natural compounds.

Efficient purification of a recombinant tag-free thermostable Kluyveromyces marxianus uricase by pH-induced self-cleavage of intein and expression in Escherichia coli

Uricase as an important healthcare-related protein is extensively used in the treatment of tumor lysis syndrome and in the manufacture of serum uric-acid diagnostic kits. In this study, a gene of a new thermostable uricase (KmUOX) was cloned from thermotolerant yeast Kluyveromyces marxianus. The uricase was fused with a self-cleaving intein and cellulose-binding affinity tag and expressed in Escherichia coli BL21 (DE3). Through the binding to inexpensive cellulose and in situ intein cleavage induced by a pH change, tag-free uricase (KmUOX) was efficiently purified with a 77.11% yield via a single-step column purification strategy. This tag-free uricase showed Km, Vmax, and Kcat values of 67.60µM, 56.35µM/(minmg), and 32.74S-1, respectively. Furthermore, this pure uricase was relatively thermostable and retained 79.75% of activity when incubated at 40°C for 90h. Thus, this pH-induced self-cleavable intein system combined with a cellulose matrix for affinity chromatography is proven here to be an effective and low-cost method for recombinant-uricase purification. Moreover, the stability of KmUOX makes it useful for clinical applications.

Preparation of Low Cost Affinity Chromatographic Matrix and Its Application in Purification of a Lectin Isolated from Mulberry Seeds

Objective: Uniform sized agarose gel beads were prepared from agarose by emulsification technique. The prepared matrix has been attached with commercially available concanavalin A (conA) and finally it has applied to purify mulberry seed lectin. The present work shows a simple and inexpensive method for the preparation of an affinity matrix for purification of conA specific mulberry seed lectin. Method: Firstly, conA was immobilized on agarose gel beads and coupled to hexadiamine by using amino reactive bifunctional crosslinker (2,4,6-trichloro-1,3,5-trizine). Then it used as affinity matrix for the purification of mulberry seed lectin (designated as MSL). In purification protocol initially conA has been immobilized with agarose. Then MSL have been purified from the seeds of Morus alba L. Result: The agarose beads showed the best uniformity when 2-2.5% concentration of Tween 80 was used. The newly prepared affinity matrix agarose gel was able to purify MSL with the molecular weight of 22 kDa in a single step. The purified lectin strongly agglutinated with mice, chicken, bovine and human blood types A, B and O erythrocytes respectively. There was no activity found on goat erythrocytes. Conclusion: The present work shows a simple and inexpensive method for the preparation of an affinity chromatographic matrix. The prepared matrix was applied to purify mulberry seed lectin.

Assessment of Affinity Chromatography Matrices in Plantibody Purification from Aqueous Two-Phase Extraction Samples

Assessment of Affinity Chromatography Matrices in Plantibody Purification from Aqueous Two-Phase Extraction Samples

<b>PVA-Glutaraldehyde as support for lectin immobilization and affinity chromatography

Immobilized lectins are a powerful biotechnological tool for separation and isolation of glycoconjugates. In the present study, polyvinyl alcohol (PVA) and glutaraldehyde (GA) were used as a support for Concanavalin A (Con A) covalent immobilization and for entrapment of Parkia pendula seed gum (PpeG). Con A immobilization yielded approximately 30% and 0.6 M glucose solution was the minimum concentration able to elute fetuin from column. PVA-GA-PpeG column was efficiently recognized by pure P. pendula lectin (PpeL). These findings indicate that PVA-GA interpenetrated network showed to be an efficient support for lectin covalent immobilization and as affinity chromatography matrix after trapping of PpeG.

Affinity chromatography matrices for depletion and purification of casein glycomacropeptide from bovine whey.

Casein glycomacropeptide (CMP) is a 64- amino acid peptide found in cheese whey, which is released after κ-casein specific cleavage by chymosin. CMP lacks aromatic amino acids, a characteristic that makes it usable as a nutritional supplement for people with phenylketonuria. CMP consists of two nonglycosylated isoforms (aCMP A and aCMP B) and its different glycosylated forms (gCMP A and gCMP B). The most predominant carbohydrate of gCMP is N-acetylneuraminic acid (sialic acid). Here, we developed a CMP purification process based on the affinity of sialic acid for wheat germ agglutinin (WGA). After formation of chitosan beads and adsorption of WGA, the agglutinin was covalently attached with glutaraldehyde. Two matrices with different WGA density were assayed for CMP adsorption. Maximum adsorption capacities were calculated according to the Langmuir model from adsorption isotherms developed at pH 7.0, being 137.0 mg/g for the matrix with the best performance. In CMP reduction from whey, maximum removal percentage was 79% (specifically 33.7% of gCMP A and B, 75.8% of aCMP A, and 93.9% of aCMP B). The CMP was recovered as an aggregate with an overall yield of 64%. Therefore, the matrices developed are promising for CMP purification from cheese whey. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:171-180, 2017.

Isolation of a new ssDNA aptamer against staphylococcal enterotoxin B based on CNBr-activated sepharose-4B affinity chromatography.

Staphylococcus aureus are potent human pathogens possessing arsenal of virulence factors. Staphylococcal food poisoning (SFP) and respiratory infections mediated by staphylococcal enterotoxin B (SEB) are common clinical manifestations. Many diagnostic techniques are based on serological detection and quantification of SEB in different food and clinical samples. Aptamers are known as new therapeutic and detection tools which are available in different ssDNA, dsDNA and protein structures. In this study, we used a new set of ssDNA aptamers against SEB. The methods used included preparation of a dsDNA library using standard SEB protein as the target analyte, affinity chromatography matrix in microfuge tubes, SELEX procedures to isolate specific ssDNA-aptamer as an affinity ligand, aptamer purification using ethanol precipitation method, affinity binding assay using ELISA, aptamer cloning and specificity test. Among 12 readable sequences, three of them were selected as the most appropriate aptamer because of their affinity and specificity to SEB. This study presents a new set of ssDNA aptamer with favorable selectivity to SEB through 12 rounds of SELEX. Selected aptamers were used to detect SEB in infected serum samples. Results showed that SEB c1 aptamer (2 µg SEB/100 nM aptamer) had favorable specificity to SEB (kd = 2.3 × 10(-11) ). In conclusion, aptamers can be considered as useful tools for detecting and evaluating SEB. The results showed that affinity chromatography was an affordable assay with acceptable accuracy to isolate sensitive and selective novel aptamers. Copyright © 2016 John Wiley & Sons, Ltd.

Proteolytic cleavage at twin arginine residues affects structural and functional transitions of lupin seed 11S storage globulin.

The 11S storage globulin of white lupin seeds binds to a metal affinity chromatography matrix. Two unusual stretches of contiguous histidine residues, reminiscent of the multiple histidines forming metal binding motifs, at the C-terminal end of 11S globulin acidic chains were hypothesized as candidate elements responsible for the binding capacity. To prove this, the protein was incubated with a lupin seed endopeptidase previously shown to cleave at twin arginine motifs, recurrent in the sequence region of interest. Upon incubation with this enzyme, the loss of metal binding capacity paralleled that of the anti-his-tag reactive polypeptides. The recovered small proteolytic fragment was analyzed by mass spectrometry and N-terminal sequencing and found to correspond to the 24-mer region cleaved off at twin arginine residues and containing the natural his-tag-like region. Similarly, when lupin seeds were germinated for a few days, the his-tag containing 11S globulin chain was converted to a form devoid of such region, suggesting that this mechanism is a part of the natural degradatory process of the protein. The hypothesis that the ordered and controlled dismantling of storage proteins may generate peptide fragments with potential functional roles in plant ontogenesis is presented and discussed.

Characterization of a Novel Agarose–Nickel Composite Matrix for Protein Nanoparticles Affinity Chromatography in Expanded Bed

The novel agarose–nickel (Ag–Ni) expanded bed matrix was investigated with regard to suitability for practical recovery of nano-bioproducts (NBPs) such as protein nanoparticles as drug delivery carriers. The matrix was immobilized by Reactive Green 19 (RG19) dye–ligand and was subjected to biochemical evaluation through batch adsorption studies (isotherm and kinetic studies) and column chromatography of bovine serum albumin nanoparticles (BSA NPs) with average size of 85–95 nm as a model system. Based on adsorption isotherm investigations, the adsorption phenomenon appeared to follow the Langmuir isotherm model with maximum binding capacity of 24.9 mg/ml adsorbent. Subsequently adsorption data were modeled using the pseudo-first-order and pseudo-second-order kinetics equation. The results demonstrated that the adsorption process kinetics followed the pseudo-first-order kinetic model. The dynamic binding capacity (DBC) for BSA NP adsorption was calculated at various flow velocities which showed favorable column efficiency at relatively high flow rates. BSA NPs recovery was studied in the expanded bed column which resulted in 74 % recovery. The results indicated that the novel resin is a promising chromatographic medium for protein nanoparticle separation with high adsorption capacity and column efficiency at reasonably high flow rates. The generic application of such dye–ligand immobilized composite matrix for the adsorption and purification of BSA NPs as a nanoparticulate bioproduct was discussed.

A comparative study of chromatographic matrices for affinity chromatography of the diphtheria toxin variant Cross-Reacting Material 197

A comparative study of chromatographic matrices for affinity chromatography of the diphtheria toxin variant Cross-Reacting Material 197

Expression of liver-targeting peptide modified recombinant human endostatin and preliminary study of its biological activities.

Recombinant human endostatin (rEndostatin or endostar) has been shown to inhibit endothelial cells proliferation, migration, and angiogenesis and exhibits a broad spectrum of activities against solid tumors. However, rEndostatin is easily degradable and evenly distributed to all tissues. Selectively delivering rEndostatin to the lesion site might be more potent. The circumsporozoite protein (CSP) coats the malarial sporozoite and targets the liver for infection; I-plus of N end of CSP could specifically bind to the liver. Based on this, we hypothesize the fusion protein with introducing the CSP I-plus sequence into rEndostatin (rES-CSP) of which not only targets the liver, but also inhibits endothelial cells proliferation, migration, and tube formation. Therefore, it selectively reduces angiogenesis of hepatocellular carcinoma (HCC) and improves the anti-HCC effect. In this study, we synthesized a novel rES-CSP fusion gene by SOE-PCR and expressed the fusion protein in Escherichia coli BL2l (DE3). The suitable conditions were optimized by an orthogonal test (L(25)(5)(4)). The yields were 12mg/l culture medium following refolding and purification on nickel-nitrilotriacetic acid (Ni-NTA) metal affinity chromatography matrices. The purified rES-CSP is specifically targeted to the hepatocyte and inhibited the proliferation and migration of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner and showed potent antiangiogenic capability on HUVECs tube formation assay and chick embryo chorioallantoic membrane (CAM) assay. These results lay the foundation for the further study of its targeting and anti-HCC in vivo and provide a feasible and convenient approach to produce liver-targeting drugs for treatment of the liver diseases.

Improved purification and enzymatic properties of a mixture of Sticholysin I and II: Isotoxins with hemolytic and phospholipase A2 activities from the sea anemone Stichodactyla helianthus

Sticholysin I and Sticholysin II (StI and StII) are two potent hemolysins which form pores in natural and model membranes at nanomolar concentrations. These proteins were purified from the aqueous extract of the sea anemone Stichodactyla helianthus, Ellis 1768, by gel filtration and ionic exchange chromatography. This procedure rendered StI and StII with high purity (purification factors: 36 and 50, respectively) but a low yield of hemolytic activity, HA (<3%). Additionally, these toxins exhibited very low phospholipase activity (10−3U/mg of protein). In this work, a mixture StI–StII was obtained (yield >95%, with an increase in specific activity: 14 times) from the animal extract using an oxidized phospholipid-based affinity chromatographic matrix binding phospholipases. Cytolysin identification in the mixture was performed by immunoblotting and N-terminal sequence analyses. Phospholipase A2 (PLA2) activity of StI–StII was relatively high (1.85U/mg) and dependent of Ca2+. The activity resulted optimum when was measured with the mostly unsaturated soybean phosphatidylcholine (PC), when compared to the less unsaturated egg PC or completely saturated dipalmitoyl PC, in the presence of 40mM Ca2+ at pH 8.0. This Ca2+ concentration did not exert any effect on binding of StI–StII with soybean PC monolayers. Then, PLA2 activity seems not be required to binding to membranes.