Covalent Coupling Of Proteins Research Articles

Environmentally friendly covalent coupling of proteins onto oxidized cellulosic materials

Cellulose is a biodegradable and renewable material that is one of the most abundant biopolymers with many different applications from low value newsprint products to high value biomedical sensor devices.

Latex particles sensitized with proteins of Leptospira interrogans for application in immunoagglutination assays

ABSTRACTPhysical adsorption and chemical coupling of recombinant proteins of Leptospira interrogans onto polystyrene and core-shell carboxylated particles were, respectively, investigated with the aim of producing latex–protein complexes to be used in immunoagglutination assays able to detect the leptospirosis disease in either humans or animals. To this effect, a protein lysate of crude extracts was evaluated, and sensitizations were carried out at different pHs, with the antigenic proteins approach to the particle surface favored at pH close to their isoelectric point. In the covalent coupling experiments, high fractions of proteins were chemically bound to carboxyl groups on the particle surface and higher densities of linked proteins were obtained for particles exhibiting greater carboxyl group densities. The produced latex–protein complexes were tested in immunoagglutination assays, by turbidimetry and a visual method, from a panel that included positive and negative bovine, canine, and human sera. The area under receiver operating characteristic curves was used as an index of accuracy. The complexes obtained by covalent coupling of proteins on the latex of higher density of carboxyl groups allowed an acceptable discrimination between the studied positive and negative sera.

Modification of ovalbumin with fructooligosaccharides: consequences for network morphology and mechanical deformation responses.

The Maillardation of proteins has been used as a natural alternative to improve its functionality by covalent coupling of proteins with saccharides. However, the impact of Maillard reaction on the structural aspects of protein networks and, as a consequence, the mechanical breakdown properties of the gel networks has not been reported. The objective of this study was to evaluate how the attachment of linear oligo-sugar moieties onto ovalbumin affects its aggregation, network morphology, and consequently the mechanical deformation properties including the ability of the networks to elastically store energy in this material. To potentially alter the morphology of the network structure, ovalbumin was modified by conjugating some of its amino groups with fructooligosaccharide (FOS) moieties via the Maillard reaction. It was demonstrated that the attachment of FOS to ovalbumin does not affect the integrity of the secondary and tertiary structure as characterized using circular dichroism and tryptophan fluorescence. Differences in the network morphology were observed by scanning electron microscopy for FOS-modified ovalbumin variants. Upon increased modification, the microstructure of the gels had more and larger pores and had thinner strands than nonmodified variants. Evaluation of the large deformation properties of the gels demonstrated that FOS-modified gels were less strong and less brittle and showed lower stiffness than nonmodified variants. The recoverable energy (elastically stored energy) of gels reduced with an increase in the degree of modification. The results show that the attachment of FOS to ovalbumin alters the structural and mechanical (large) breakdown properties of the protein gels. The consequences of the alteration of the network structure and large deformation properties of FOS-modified ovalbumin offer opportunities to efficiently design food materials with desirable techno-functional applications.

Detailed AFM Force Spectroscopy of the Interaction Between CD44–IgG Fusion Protein and Hyaluronan

Atomic force microscopy (AFM) force spectroscopy was used to study the single-molecule rupture events of the interaction between hyaluronan (HA) and the binding domain of its cell surface receptor CD44. AFM probes were amino terminated with 3-aminopropyl triethoxy silane (APTES) followed by covalent coupling of protein A, enabling the binding of the CD44–HA-binding domain, as part of a CD44–Fc fusion protein. HA was covalently bound to APTES-coated silicon surfaces. Single-rupture events were recorded at various loading rates revealing an energy barrier: Eb = 24 ± 1 kT and characteristic distance: xβ = 1.3 ± 0.1 nm for this interaction. This quantification will be of interest in applications and research involving the use of the CD44–Fc fusion protein since we observe a weaker interaction between HA and CD44–Fc than what has been reported for the entire native CD44 molecule.

Specific labelling of cell populations in blood with targeted immuno-fluorescent/magnetic glyconanoparticles

Current performance of iron oxide nanoparticle-based contrast agents in clinical use is based on the unspecific accumulation of the probes in certain organs or tissues. Specific targeted biofunctional nanoparticles would significantly increase their potential as diagnostic and therapeutic tools in vivo. In this study, multimodal fluorescent/magnetic glyco-nanoparticles were synthesized from gold-coated magnetite (glyco-ferrites) and converted into specific probes by the covalent coupling of protein G and subsequent incubation with an IgG antibody. The immuno-magnetic-fluorescent nanoparticles were applied to the specific labelling of peripheral blood mononuclear cells (PBMCs) in a complex biological medium, as human blood. We have been able to label specifically PBMCs present in blood in a percentage as low as 0.10–0.17%. Red blood cells (RBCs) were also clearly labelled, even though the inherent T 2 contrast arising from the high iron content of these cells (coming mainly from haemoglobin). The labelling was further assessed at cellular level by fluorescence microscopy. In conclusion, we have developed new contrast agents able to label specifically a cell population under adverse biological conditions (low abundance, low intrinsic T 2, high protein content). These findings open the door to the application of these probes for the labelling and tracking of endogenous cell populations like metastatic cancer cells, or progenitor stem cells that exist in very low amount in vivo.

The use of bifunctional polyethyleneglycol derivatives for coupling of proteins to and cross-linking of collagen matrices.

The realization of three-dimensional (3D) degradable matrices which slowly release bio-active components represents a major challenge in the field of tissue engineering. In this paper we report on the usage of commercially available bifunctional agents for both the covalent coupling of proteins to and the cross-linking of collagen matrices. Proteins - horse radish peroxidase (HRP) was used as a model protein - were cross-linked with either a homobifunctional (disuccinimidyldisuccinatepolyethylene-glycol) or a heterobifunctional (N-hydroxysuccinimidylvinylsulfonepolyethyleneglycol) agent. In the case of the heterobifunctional cross-linking agent the collagen matrices were previously modified with succinimidylacetylthioacetate in order to introduce sulfhydryl groups. As compared with control experiments a 10-fold and 50-fold increase of immobilized proteins were achieved with the homobifunctional and heterobifunctional cross-linker resp. The HRP-PEG conjugates demonstrated a better long-term stability as compared to the non-treated HRP. The effects of the cross-linking agents and the thiolation reagent succinimidylacetylthio acetate on the in vitro degradation of the collagen matrices by collagenase were also investigated. In particular the reaction with succinimidylacetylthio acetate appears to offer interesting opportunities both for coupling active proteins and modulating the degradation times of collagen matrices.

Crosslinked, hydrophilic, azlactone-functional polymeric beads: A two-step approach

Crosslinked, hydrophilic, azlactone-functional polymeric beads are readily prepared via a two-step approach involving: (1) reverse phase suspension copolymerization of N-acryloyl-amino acids with water-soluble crosslinkers and, optionally, dimethylacrylamide, followed by (2) cyclodehydration of pendant acylamino acid groups to azlactones using acetic anhydride. Azlactone functionalities of 0.3 to 3.0 meq/g (typically greater than 70% of the theoretical value) were achieved by this procedure. The azlactone-functional group in these beads was found to be quite reactive towards amine nucleophiles, even in aqueous solution where little competition from hydrolysis was observed. Rapid, covalent coupling of protein could be accomplished from aqueous media under mild conditions, and indicated a potential for extremely high coupling densities (up to 245 mg protein/g of beads).

Interaction of creatine kinase and hexokinase with the mitochondrial membranes, and self-association of creatine kinase: crosslinking studies.

Covalent coupling of protein by crosslinking reagents have been used to study the interaction of mitochondrial creatine kinase (CKm) and hexokinase (HK) with the mitochondrial membranes. The effects of crosslinkers were studied either by following the inhibition of solubilization of enzymatic activities or by modification of the electrophoretic patterns of proteins solubilized from mitochondria after treatment with different crosslinkers. Dimethylsuberimidate (DMS) efficiently reduced the amount of HK activity solubilized by various agents but it did not modify solubilization of CKm from mitochondria. The effect of DMS on HK solubilization did not result from non specific crosslinking since it did not impede the solubilization of adenylate kinase. Bissuccinimidyl another class of crosslinker has been tested. Ethyleneglycol bis (succinimidyl succinate)(EGS) efficiently reduced HK solubilization, but in addition it induced osmotic stabilization of mitochondria and thus impeded release of soluble or solubilized proteins from the intermembrane space. Furthermore this agent drastically inhibited CKm activity and thus, in a second set of experiments the effect of crosslinkers have been studied by the disappearance of protein bands in the electrophoretic pattern of soluble fractions obtained from mitochondria, the outer membranes of which have been ruptured to allow free release of soluble proteins. Results of these experiments showed that succinimidyl reagents and Cu++-Phenanthroline substantially reduced the amount of CKm released from mitochondria and confirmed that bisimidates were ineffective in inhibiting CKm solubilization. In addition crosslinking reagents have been used to study subunits interactions in purified CKm. Our results showed, in contrast with control experiments with a non oligomeric protein (ovalbumin) which did not give rise to polymers, that in the same conditions electrophoresis of crosslinked CKm resolved a set of species with molecular weights roughly equal to integral multiples of the protomer. These results proved that the polymeric form of CKm was an octamer.

Comparison between adsorption and covalent coupling of proteins to solid phases using different polymers as support

Comparison between adsorption and covalent coupling of proteins to solid phases using different polymers as support