Comparison of separation modes for microchip electrophoresis of proteins.

Separation of a set of model proteins was tested on a microchip electrophoresis analytical platform capable of sample injection by two different electrokinetic mechanisms. A range of separation modes-microchip capillary zone electrophoresis, microchip micellar electrokinetic chromatography, and nanoparticle-based sieving-was tested on glass and polydimethylsiloxane/glass microchips and with silica-nanoparticle colloidal arrays. The model proteins calmodulin (18 kiloDalton), bovine serum albumin (66 kDa), and concanavalin (106 kDa) were labeled with Alexa Fluor 647 for laser-induced fluorescence detection. The best separation and resolution were obtained in a silica-nanoparticle colloidal array chip.

Biopolymer monolith for protein purification.

Porous glycopolymers, "glycomonoliths", were prepared by radical polymerization based on polymerization-induced phase separation with an acrylamide derivative of α-mannose, acrylamide and cross-linker in order to investigate protein adsorption and separation. The porous structure was induced by a porogenic alcohol. The pore diameter and surface area were controlled by the type of alcohol. The protein adsorption was measured in both batch and continuous flow systems. The glycomonoliths showed specific interaction with the sugar recognition protein of concanavalin A, and non-specific interaction to other proteins was negligible. The amount of protein adsorption to the materials was determined by the sugar density and the composition of the glycomonoliths. Fundamental knowledge regarding the glycomonoliths for protein separation was obtained.

ConA-Like Lectins: High Similarity Proteins as Models to Study Structure/Biological Activities Relationships.

Lectins are a widely studied group of proteins capable of specific and reversible binding to carbohydrates. Undoubtedly, the best characterized are those extracted from plants of the Leguminosae family. Inside this group of proteins, those from the Diocleinae subtribe have attracted attention, in particular Concanavalin A (ConA), the best-studied lectin of the group. Diocleinae lectins, also called ConA-like lectins, present a high similarity of sequence and three-dimensional structure and are known to present inflammatory, vasoactive, antibiotic, immunomodulatory and antitumor activities, among others. This high similarity of lectins inside the ConA-like group makes it possible to use them to study structure/biological activity relationships by the variability of both carbohydrate specificity and biological activities results. It is in this context the following review aims to summarize the most recent data on the biochemical and structural properties, as well as biological activities, of ConA-like lectins and the use of these lectins as models to study structure/biological activity relationships.

Disulphide production by Ero1α-PDI relay is rapid and effectively regulated.

The molecular networks that control endoplasmic reticulum (ER) redox conditions in mammalian cells are incompletely understood. Here, we show that after reductive challenge the ER steady-state disulphide content is restored on a time scale of seconds. Both the oxidase Ero1α and the oxidoreductase protein disulphide isomerase (PDI) strongly contribute to the rapid recovery kinetics, but experiments in ERO1-deficient cells indicate the existence of parallel pathways for disulphide generation. We find PDI to be the main substrate of Ero1α, and mixed-disulphide complexes of Ero1 primarily form with PDI, to a lesser extent with the PDI-family members ERp57 and ERp72, but are not detectable with another homologue TMX3. We also show for the first time that the oxidation level of PDIs and glutathione is precisely regulated. Apparently, this is achieved neither through ER import of thiols nor by transport of disulphides to the Golgi apparatus. Instead, our data suggest that a dynamic equilibrium between Ero1- and glutathione disulphide-mediated oxidation of PDIs constitutes an important element of ER redox homeostasis.

Surface-imprinted silica particles for Concanavalin A purification from Canavalia ensiformis.

Concanavalin A is a representative of the plant protein group known as lectins. Many lectin proteins have useful characteristics for studies on cell division and cell surfaces. In this study, a new adsorbent for the specific separation of Concanavalin A was prepared by applying a silica particle surface imprinting method. First, silica particles were activated via acidic treatment, and then, 3-methacryloyloxypropyl trimethoxysilane (MPTMS) was used for modification. For the preparation of Concanavalin A surface-imprinted silica particles (Con A-MISPs), N-methacryloyl-l-histidine methyl ester (MAH) was used as a functional monomer. The silica particles were characterized using a Zetasizer, scanning electron microscopy equipment (SEM), and Fourier transform infrared spectroscopy (FTIR). The effects of parameters such as the pH, initial concentration of Concanavalin A, and temperature on the adsorption of Concanavalin A were determined. The maximum Concanavalin A adsorption onto Con A-MISPs was observed to be 305.2 mg/g at a pH of 6. The reusability of the Con A-MISPs was approximately 93.5%. The non-imprinted silica particles (NISPs) were prepared in the same manner without Concanavalin A to compare the surface imprinting factor. Selective binding studies were carried out with lysozyme and hemoglobin molecules. The selectivity of the Con A-MISPs was also investigated by isolating Concanavalin A from Canavalia ensiformis. The purity of the Concanavalin A was shown by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).

ISFET and Dex-AgNPs based portable sensor for reusable and real-time determinations of concanavalin A and glucose on smartphone.

In this paper, a portable real-time sensing device was built for Concanavalin A (Con A) and glucose detection using a smartphone. The ion-sensitive field-effect transistor (ISFET) functioning at a low working point was selected as a small-size, low-power transducer, and dextran-capped silver nanoparticles (Dex-AgNPs) served as sensitive nanoprobes on the ISFET gate. Using the affinity between Con A and carbohydrates, Con A can be captured, and thus directly detected by the ISFET/Dex-AgNPs unit; then glucose can be determined indirectly by removing Con A from the ISFET/Dex-AgNPs/Con A unit via competition with dextran. The mechanism of this competition does less harm to the sensor, allows the reusability of the sensing device, and overcomes the Debye screening of the FET device in saline solutions. Powered by a button cell, the handheld device attains excellent Con A (0.16 ng mL-1) and glucose (10 nM) detection limit, and can practically be used for at least 20 days.

Ferromagnetic levan composite: an affinity matrix to purify lectin.

A simple and inexpensive procedure used magnetite and levan to synthesize a composite recovered by a magnetic field. Lectins from Canavalia ensiformis (Con A) and Cratylia mollis (Cramoll 1 and Cramoll 1, 4) did bind specifically to composite. The magnetic property of derivative favored washing out contaminating proteins and recovery of pure lectins with glucose elution. Cramoll 1 was purified by this affinity binding procedure in two steps instead of a previous three-step protocol with ammonium sulfate fractionation, affinity chromatography on Sephadex G-75, and ion exchange chromatography through a CM-cellulose column.

Electrochemiluminescence resonance energy transfer biosensor between the glucose functionalized MnO2 and g-C3N4 nanocomposites for ultrasensitive detection of concanavalin A.

An electrochemiluminescence (ECL) analytical platform was initially proposed based on the electrochemiluminescence resonance energy transfer (ECL-RET) mechanism for ultrasensitive detection of Concanavalin A (Con A). In this protocol, the glucose functionalized carboxylic g-C3N4 nanosheets (g-C3N4-COOH@Glu) and MnO2 nanoparticles covered carboxylic multi-wall carbon nanotubes (BSA@MnO2-MWCNTs-COOH@Glu) were synthesized and acted as ECL-RET electron donor and acceptor, respectively. Herein, glucose was served as the recognition element for binding Con A and MWCNTs was utilized as the carrier materials for loading MnO2. When the quenching probe BSA@MnO2-MWCNTs-COOH@Glu was incubated onto the modified electrodes via the specific carbohydrate-Con A interaction, the ECL signals of g-C3N4-COOH@Glu which used S2O82- as its coreactant have drastically declined. Under optimum conditions, this biosensor performed a sensitive detection of the Con A ranging from 1 × 10-5 to 1 × 104 ng/mL with a detection limit of 2.2 fg/mL (S/N = 3). Moreover, favorable analytical outcomes for detecion Con A in actual serum samples were obtained, exhibiting huge applications in clinical diagnosis of this assay.

Structural characterization of a water-soluble polysaccharide from the roots of Codonopsis pilosula and its immunity activity.

The water-soluble polysaccharide (CPP), with a molecular mass of 1.1x10(4) Da, was obtained from the roots of Codonopsis pilosula. Structure feature investigation by a combination of chemical and instrumental analysis revealed that CPP had a backbone consisting of (1-->3)-linked-beta-D-galactopyranosyl, (1-->2, 3)-linked-beta-D-galactopyranosyl and (1-->3)-linked-alpha-D-rhamnopyranosyl residues, which were branched with two glycosyl residues composed of alpha-L-arabinose-(1-->5)-alpha-L-arabinose(1-->linked residues at the O-2 position of galactosyl along the main chain in the ratio of 1:1:2:1:1. Preliminary immunological tests in vitro showed CPP could stimulate concanavalin A (ConA)- or lipopolysaccharide (LPS)-induced lymphocyte proliferation in a dose-dependent manner.

Proximity ligation detection of lectin Concanavalin A and fluorescence imaging cancer cells using carbohydrate functionalized DNA-silver nanocluster probes.

A novel method for study the interaction between carbohydrate and lectin was developed in homogenous solution. DNA was introduced to the recognition process of carbohydrate with protein. Due to the versatile detection mode of DNA, the detection efficiency for carbohydrate and protein was highly promoted. Herein, two kinds of mannose-DNA conjugates were synthesized. One of which was used to prepare DNA-templated silver nanocluster (AgNCs) and the other one contained guanine-rich DNA sequences. When the mannose of two conjugates binding to lectin Concanavalin A (Con A), great enhancement on fluorescence intensity was obtained due to the proximity ligation of the DNA-templated Ag NCs with guanine-rich DNA sequences. Hence Con A can be quantified conveniently in homogenous solution using Ag NCs with low toxicity and tunable fluorescence. Moreover, carbohydrate functionalized DNA-templated AgNCs was also utilized for cancer cell imaging based on the recognition of mannose with mannose receptor on cancer cell MDA-MB-231.

Anti-glioma properties of DVL, a lectin purified from Dioclea violacea.

Plant lectins have been studied owing to their structural properties and biological effects that include agglutinating activity, antidepressant-like effect and antitumor property. The results from this work showed the effects of the lectin extracted from the Dioclea violacea plant (DVL) on the C6 rat glioma cell line. DVL treatment was able to induce caspase-3 activation, apoptotic cell death and cellular membrane damage. Furthermore, DVL decreased mitochondrial membrane potential and increased the number of acidic vesicles and cleavage of LC3, indicating activation of autophagic processes. DVL also significantly inhibited cell migration. Compared to ConA, a well-studied lectin extracted from Canavalia ensiformes seeds, some effects of DVL were more potent, including decreasing C6 glioma cell viability and migration ability. Taken together, the results suggest that DVL can induce glioma cell death, autophagy and inhibition of cell migration, displaying potential anti-glioma activity.

Heterometal-Coordinated Monomeric Concanavalin A at pH 7.5 from Canavalia ensiformis.

The structure of concanavalin A (ConA) has been studied intensively owing to its specific interactions with carbohydrates and its heterometal (Ca²âº and Mn²âº) coordination. Most structures from X-ray crystallography have shown ConA as a dimer or tetramer, because the complex formation requires specific crystallization conditions. Here, we reported the monomeric structure of ConA with a resolution of 1.6 Å, which revealed that metal coordination could trigger sugar-binding ability. The calcium coordination residue, Asn14, changed the orientation of carbohydrate-binding residues and biophysical details, including structural information, providing valuable clues for the development and application of detection kits using ConA.

Polar silica-based stationary phases. Part III- Neutral silica stationary phase with surface bound maltose for affinity chromatography at reduced non-specific interactions.

This research article reports the coating of large pore silica microparticles with a maltose layer to which bioaffinity ligands were attached via reductive amination reaction between the aldehyde activated maltose and the amino groups of the bioaffinity ligands. This was achieved first by the periodate oxidation of the maltose-silica (MALT-silica) yielding pairs of aldehyde groups at each monosaccharide ring. These di-aldehyde functionalities were then reacted with the primary amino groups of protein bio-affinity ligands and eventually formed Schiff bases (i.e., aldimines) which were reduced using the mild reducing agent sodium cyanoborohydride to form stable amine linkages between the immobilized protein ligands and the maltose layer. Anti-human serum albumin antibody (aHSA), anti-human serum transferrin antibody (aTf) and concanavalin A (Con A) were the bio-affinity ligands immobilized onto the MALT-silica and were evaluated in high performance affinity chromatography (HPAC), namely immunoaffinity chromatography (IAC) and lectin affinity chromatography (LAC). Our initial studies reported here revealed zero or reduced nonspecific interactions with the two immunoaffinity sorbents (i.e., aHSA-MALT-silica and aTf-MALT-silica) and the lectin affinity sorbent (i.e., Con A-MALT-silica). The absence of nonspecific interactions is attributed to the hydrophilicity of the maltose layer and its shielding effect of the residual silanols (i.e., unreacted silanols) on the silica surface. Conversely, the IAC and LAC sorbents exhibited specific interactions with the target biomolecules, namely human serum albumin (HSA) and transferrin (Tf) in the case of aHSA-MALT-silica and aTf-MALT-silica columns, respectively, and glycoproteins known for their affinity to Con A in the case of Con A-MALT-silica column.

Single step purification of concanavalin A (Con A) and bio-sugar production from jack bean using glucosylated magnetic nano matrix.

Jack bean (JB, Canavalia ensiformis) is the source of bio-based products, such as proteins and bio-sugars that contribute to modern molecular biology and biomedical research. In this study, the use of jack bean was evaluated as a source for concanavalin A (Con A) and bio-sugar production. A novel method for purifying Con A from JBs was successfully developed using a glucosylated magnetic nano matrix (GMNM) as a physical support, which facilitated easy separation and purification of Con A. In addition, the enzymatic conversion rate of 2% (w/v) Con A extracted residue to bio-sugar was 98.4%. Therefore, this new approach for the production of Con A and bio-sugar is potentially useful for obtaining bio-based products from jack bean.

A high performance liquid chromatography assay for the rapid analysis of the subunit content of concanavalin A.

A high performance liquid chromatography system is described which provides a rapid and convenient assay for the relative amounts of intact (26000 dalton) and fragmented (14000 and 12000 dalton) subunits present in preparations of concanavalin A. Analyses were performed on an HPLC size exclusion column using either 8M urea or 6M guanidine hydrochloride as denaturing eluents. The efficiency and resolving power of this technique were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This HPLC assay facilitated the monitoring of the purification of concanavalin A to prepare a homogeneous preparation necessary for its biological evaluation.

Studies on lectins. XXXVI. Properties of some lectins prepared by affinity chromatography on O-glycosyl polyacrylamide gels.

A number of lectins has been purified by affinity chromatography on O-glycosyl polyacrylamide gels. The lectins isolated (and the particular sugar ligands used in the affinity carriers) are as follows: Anguilla anguilla, serum (alpha-L-fucosyl-), Vicia cracca, seeds; Phaseolus lunatus, seeds; Glycine soja, seeds; Dolichos biflorus, seeds; Maclura pomifera, seeds; Sarothamnus scoparius, seeds; Helix pomatia, ablumin glands; Clitocybe nebularis, fruiting bodies (all N-acetyl-alpha-D-galactosaminyl-); Ricinus communis, seeds (beta-lactosyl-); Ononis spinosa, root; Fomes fomentarius, fruiting bodies; Marasmius oreades, fruiting bodies (all alpha-D-galactosyl-), Canavalia ensiformis, seeds, (i.e., concanavalin A) (alpha-D-glucosyl-). Physicochemical properties of Glycine soja, Dolichos biflorus, Phaseolus lunatus, Helix Pomatia and Ricinus communis lectins corresponded well to properties of the preparations studied earlier by other workers. For the other purified lectins the essential physiochemical data (sedimentation coefficient, molecular weight, subunit composition, electrophoretic patterns, amino acid composition, carbohydrate content, isoelectric point) were established and their precipitating, hemagglutinating and mitogenic activities determined.

Induction of suppressor cells, interleukin-2 production and mitogenesis with monomeric concanavalin A: different actions of tetrameric and monomeric concanavalin A.

The ability of a photoalkylated monomeric concanavalin A (Con A) derivative to induce mitogenesis, interleukin-2 (IL-2) production and suppressor cells in murine spleen cell cultures has been compared with the activity of native, tetrameric Con A. The monomeric derivative was prepared by photochemically induced alkylation of tryptophan residues of tetravalent Con A in the presence of chloroacetamide followed by sizing chromatography [Tanaka et al. (1981) J. Biochem. 89, 1643-1646]. The monomeric derivative appeared to display less mitogenic activity than the tetramer and was also less effective in inducing IL-2 production. No difference was detected between the monomeric and tetrameric forms of Con A in inducing suppressor cells. The data suggest that cross-linking and bridging via sugar-binding sites, while potentiating mitogenesis and IL-2 production, had little effect on suppressor cell induction.

Development of an aptamer-affinity chromatography for efficient single step purification of Concanavalin A from Canavalia ensiformis.

Herein, an aptamer-based affinity chromatography method for rapid and single step purification of Concanavalin A is developed and validated. We have used a 41ntssDNA aptamer of Con A (Con A aptabody) as an affinity reagent in the developed aptamer-affinity chromatography. Stationary phase of the method consists of surface functionalized agarose beads carrying covalently immobilized Con A-aptabody. Affinity purification of Con A from jack bean (Canavalia ensiformis) seed using developed aptamer-affinity columns has resulted in ≥66% recovery with 90% purity and 336-fold purification of Con A. The developed aptamer-affinity chromatography has shown efficient scalability and consistent purification when analysed over 13mm, 20mm and 25mm diameter columns having a bed height of 60mm each. Also, the developed aptamer-agarose columns were found to be reusable with recovery decrease of 12.9% in seven sequential cycles of purification. Therefore, the developed aptamer-affinity chromatography provides a novel, efficient and single-step methodology for isolation and purification of Con A.

Structure of the gene encoding concanavalin A from Canavalia gladiata and its expression in Escherichia coli cells.

We have cloned and sequenced the gene encoding concanavalin A (Con A) from Canavalia gladiata. The sequence covers the whole transcribed region as well as the 5'- and 3'-untranscribed sequences. The coding sequence lacks introns. Con A expressed in Escherichia coli cells was purified by Sephadex G-50 affinity chromatography. The precursor of Con A expressed in E. coli undergoes a peptide cleavage and ligation in the same way as that synthesized during seed maturation.

In vitro growth of murine T cells. III. Method for separation of T cell growth factor (TCGF) from concanavalin A and biological activity of the resulting TCGF.

T cell growth factor (TCGF) resulting from the incubation of murine splenocytes with concanavalin A (Con A) has been partially purified and separated from Con A. Sequential application of 50-70% saturated ammonium sulfate precipitation and G-100 gel filtration chromatography has resulted in a 300-fold purification of TCGF with a 60% yield. 99.99% of Con A has been removed from the TCGF by these steps. TCGF exists in two molecular weight forms of about 50,000 and 25,000 daltons. TCGF activity is degradable by trypsin digestion and is stable at 56 degrees C for 30 min, but is inactivated by heating at 80 degrees C. Lymphoid cells activated by either Con A or allogeneic in vitro sensitization will grow in TCGF free of Con A but fresh splenocytes will not grow in the absence of Con A, implying a need for prior activation before TCGF sustained growth of T cells can be achieved.