Biomedical subjects

Galaev IYu

Publications and source records attributed to Galaev IYu.

10 recordsLinked to original sources

PubMed1998

Affinity precipitation of proteins: design criteria for an efficient polymer.

Affinity precipitation is fast emerging as a successful technique for the purification of proteins which can be introduced at an early stage of downstream processing. The technique applies the use of reversibly soluble-insoluble polymers which have either natural or synthetic origin. Apart from the successful use of some natural polymers, such as chitosan and alginate, the vast application of the technique depends upon the design of efficient synthetic polymers. In this laboratory, N-isopropylacrylamide (NIPAM) copolymers have been developed for metal chelate affinity precipitation of proteins. The copolymers of 1-vinylimidazole (VI) and iminodiacetic acid (IDA) with NIPAM were synthesized. The copolymers were thoroughly characterized with a view to designing an efficient soluble-insoluble polymer for metal chelate affinity precipitation of proteins.

Acrylic Resins↗

PubMed1998

Protein displacement in dye-ligand chromatography using neutral and charged polymers.

Displacement chromatography was demonstrated to perform separations efficiently under mass-overloaded conditions, offering advantages such as increased product recovery and purity, superior resolving power, and concentration and purification in a single processing step. The use of water-soluble polymers for protein displacement in dye-ligand chromatography was initiated in our laboratory. The polymers for displacement were selected using differences spectroscopy to monitor their interactions with a dye-ligand in solution. Non-charged polymers such as poly(N-vinyl pyrrolidone) and poly(N-vinyl caprolactam) efficiently displaced lactate dehydrogenase from porcine muscle from a Blue Sepahrose column. The latter polymer, being thermosensitive, could be easily removed from the eluate and recovered by precipitation at 45 degrees C and low-speed centrifugation. The positively charged polymer poly(ethylene imine) proved to be an even more efficient displacer. The dye-ligand column could be regenerated after application of displacer either by washing with a solution of the soluble ligand Cibacron Blue (in the case of non-charged polymers) or by washing with highly alkaline solutions containing polyanions (in the case of poly(ethylene imine)) The latter formed a soluble complex with poly(ethylene imine) and stripped the column from the polymer.

Animals↗

PubMed1998

Reactivation of glyceraldehyde-3-phosphate dehydrogenase using conjugates of monoclonal antibodies with polyelectrolyte complexes. An attempt to make an artificial chaperone.

The simplified model of chaperone action when the inactive misfolded forms are removed from the reaction media preventing aggregation was developed using antibodies in combination with polyelectrolyte complexes. The antibodies, which bind specifically inactive dimers of glyceraldehyde-3-phosphate dehydrogenase but not native tetramers, were coupled covalently to poly(methacrylic acid). The treatment of inactivated GAPDH with this conjugate followed by its precipitation after equimolar addition of polycation, poly-(N-ethyl-4-vinylpyridinium bromide), resulted in a significant increase in the specific activity of the enzyme.

Animals↗

PubMed1996

Polymer-shielded dye-ligand chromatography of lactate dehydrogenase from porcine muscle in an expanded bed system.

Dye-affinity chromatography is a widely used technique in protein purification. It has recently been shown that the efficiency of the chromatography process can be significantly improved by pretreatment of the affinity matrix with certain water soluble polymers such as poly(vinyl pyrrolidone). This technique termed as polymer-shielded, dye-affinity chromatography has been successfully used in packed bed mode at a lab scale for the purification of a number of enzymes. The present work deals with the application of polymer-shielded dye-affinity chromatography in an expanded bed system of Streamline-Cibacron Blue 3GA for the isolation of lactate dehydrogenase from a crude porcine muscle extract. The elution conditions were optimised to obtain an efficient process. A higher recovery of the target enzyme (78%) was obtained from the polymer shielded column as compared to the unshielded column (17%), after low ionic strength elution. The purification factor obtained after chromatography on the polymer shielded column was higher (4.1) than that from unshielded column (1.8).

Animals↗

PubMed1993

Thermoreactive water-soluble polymers, nonionic surfactants, and hydrogels as reagents in biotechnology.

Thermoprecipitating polymers such as poly (N-isopropylacrylamide), poly(N-vinyl caprolactam), and some ethylene oxide-containing surfactants appear to be suitable for developing new separation systems to complement traditional precipitation, chromatography, and extraction of biological molecules. The nature of thermally induced phase separation of polymers and nonionic surfactants is discussed and examples are given. Covalent coupling of an enzyme to a thermoprecipitable polymer results in a biocatalyst which combines the qualities of soluble and immobilized enzymes. Biocatalysts of this type can be separated from reaction media by precipitation after temperature increase. The use of thermoprecipitating polymer-protein conjugates in immunoassays overcomes one of the shortcomings of traditional methods with solid sorbent-linked antigen or antibody-diffusional limitations. Thermoreactive hydrogels produced by crosslinking of thermoprecipitating polymers can be successfully used for concentrating macromolecules or microbe-rich slurries. Alternate volume changes of hydrogels on heating and cooling produce a "hydraulic pump" which can enhance the productivity of an immobilized biocatalyst. Hydrogels could be used to control reaction or diffusion rates by a thermal feedback mechanism.

Biotechnology↗

PubMed1982

A kinetic study of hog kidney aminoacylase.

The kinetic and thermodynamic parameters of the hog kidney acylase-catalyzed reactions of N-acetyl-L-methionine hydrolysis and synthesis have been investigated. The equilibrium constants were determined at high concentrations of the products (acetate and L-amino acid) for a number of amino acids. A kinetic scheme of the enzymatic reaction was proposed that describes the dependence of the rate of hydrolytic and synthetic reactions on the composition of the reaction system. The kinetic parameters determined from the progress curves proved very close to those obtained by the initial rate analysis. The kinetic and thermodynamic constants fitted the Haldane equation.

Amidohydrolases↗

PubMedDate not supplied

Dye-affinity techniques for bioprocessing: recent developments.

Textile or triazine dyes play an important role as affinity ligands in protein purification. Each step of the protein purification protocol can be divided into three stages, partitioning between two phases, separation of these phases and recovery of the target protein from the enriched phase. Now developments in dye-affinity techniques are discussed emphasizing the innovations in all three stages of the protein purification process. Dye-affinity chromatography has become a routine step in protein purification. New dyes have been developed and used successfully in both traditional chromatographic mode and new modes like affinity precipitation, polymer aqueous two-phase partitioning or expanded bed chromatography. The specificity of dye techniques has been increased by both purposeful designing of new dyes and decreasing non-specific protein-dye interactions with polymer shielding. One can envisage further development and ramification of dye-affinity techniques in protein purification.

Affinity Labels↗

PubMedDate not supplied

Polymer-shielded dye-affinity chromatography.

Polymer-shielded dye-affinity chromatography is a form of chromatography in which the dye matrix forms complexes with a nonionic, water-soluble polymer such as poly(vinylpyrrolidone) or poly(vinyl alcohol), prior to the column chromatography of a crude protein extract, the idea being that polymer shielding of the dye will prevent nonspecific interactions between the target protein and the dye. The concept of polymer shielding and a strategy for the rational selection of polymers suitable as shielding agents are presented.

Animals↗

PubMedDate not supplied

Polycomplexes--potential for bioseparation.

This review discusses the properties of complexes formed by proteins with polyelectrolytes (PPC) and two polyelectrolyte molecules of opposite charge (PEC). The most highly charged polymers with ionic groups in each monomer unit are considered in this paper. There are all reasons to regard PEC as macromolecular compounds produced as a result of equilibrium reactions with inherent permanent exchange of polyions in water-salt solutions. They combine two properties that might appear at first sight to be mutually exclusive, i.e. rather high stability and lability. Introduction of bioaffinity ligands endows PEC with the recognition capacity sufficient for the purposes of bioseparation and bioanalysis. Antibody-PEC conjugates were successfully used in the immunoassay combining the advantages of both homogeneous and heterogeneous assays and for modeling of chaperone action. The unique properties of polyelectrolyte complexes in combination with bioaffinity ligands makes them promising for the development of highly efficient means of protein isolation, new immunoassay procedures and creation of reversibly soluble biocatalysts.

Electrolytes↗

PubMedDate not supplied

Affinity precipitation of monoclonal antibodies by nonstoichiometric polyelectrolyte complexes.

The nonstoichiometric polyelectrolyte complex (PEC) formed by poly(methacrylic acid) (degree of polymerization 1830) (PMAA) and poly(N-ethyl-4-vinyl-pyridinium bromide) (degree of polymerization 530) (PEVP) undergoes reversible precipitation from aqueous solution at any desired pH-value in the range 4.5-6.5 depending on the ionic strength and PEVP/PMAA ratio in the complex. The antigen, inactivated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rabbit was covalently coupled to PEVP. The resulting GAPDH-PEVP/PMAA complex was used for the purification of antibodies from a 6G7 clone specific towards inactivated GAPDH. The crude extract was incubated with GAPDH-containing PEC and the precipitation of the PEC was carried out at 0.01 M NaCl and pH 4.5, 5.3, 6.0 and 6.5 using PEC with PEVP/PMAA ratios of 0.45, 0.3, 0.2 and 0.15, respectively. Purified antibodies were eluted at pH 4.0 where PECs of all compositions used were insoluble. PEC precipitation is accompanied only by small nonspecific coprecipitation of proteins. Precipitated PEC could be dissolved at pH 7.3 and used repeatedly.

Animals↗