Isolation of microbial DNA by newly designed magnetic particles.

Carboxyl group-containing magnetic nonporous poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) (P(HEMA-co-GMA)) microspheres and cobalt ferrite nanoparticles modified with alginic acid (natural carboxylic polysaccharide) were used for isolation of microbial DNA of lactic acid bacteria (LAB) from dairy products, lyophilised cell cultures, and bacterial colonies grown on hard media, and Trichophyton fungi DNA from lyophilised cells. DNA from the samples with lysed cells was reversibly adsorbed to the particles in the presence of high poly(ethylene glycol) (PEG 6000) and sodium chloride concentrations. The optimal final PEG and NaCl concentrations were 9.1 wt.% and 2.0 M, respectively. The adsorbed DNA was released from the particles in low ionic strength TE buffer. The quality of isolated DNA was checked by PCR amplification. Moreover, PCR amplicons were isolated on cobalt ferrite nanoparticles modified with alginic acid and checked by restriction analysis.

Immunoaffinity isolation of CEACAM1 on hydrazide-derivatized cellulose with immobilized monoclonal anti-CEA antibody.

Carcinoembryonic cell adhesion molecule 1 (CEACAM1) is a human membrane glycoprotein belonging to the carcinoembryonic antigen (CEA) family and to the immunoglobulin superfamily. It is expressed in apical membranes of many epithelial cells in gastrointestinal and urogenital tract and also in granulocytes and lymphocytes, and its biological effect in human tissues has recently been discussed in literature. The purpose of this study was to isolate CEACAM1 glycoprotein from bile and characterize its purity and recovery which has not been described before. Affinity chromatography of CEACAM1 on hydrazide-activated cellulose with immobilized monoclonal anti-CEA F34-187 antibody is described. The immunoglobulin carbohydrate moiety was oxidized by periodate and then bound to hydrazide-activated matrix. Crude protein fraction from bile was applied on the affinity column and after extensive washing of non-bound proteins CEACAM1 was eluted with 6 M guanidine-HCl. A single immunopositive 85 kDa band was detected on Western blots with anti-CEA antibody after SDS-PAGE. We found out that CEACAM1 was not stainable with any common method of protein staining and the only non-specific method which could detect the 85 kDa band was a lectin staining.

Properties of RNase A immobilized on magnetic Poly(2-hydroxyethyl methacrylate) microspheres.

Magnetic hydrogel microspheres 1.5 microm in size were prepared by dispersion copolymerization of 2-hydroxyethyl methacrylate and ethylene dimethacrylate in the presence of magnetite, which formed the core of the particles. RNase A was coupled to the particles by the cyanuric chloride method. Gel electrophoresis of plasmid DNA pUC 19 (contaminated by bacterial RNA) confirmed RNA degradation with the immobilized enzyme. The effect of temperature and pH on the relative activity of immobilized RNase A was estimated after incubation of the samples at different temperatures (30-80 degrees C) and pH (4.0-8.0). Maximum relative activity was observed at 70 degrees C and pH 6.5. The matrices based on magnetic poly(HEMA) had a low tendency to adsorb RNA.

Detection of pathological changes of proteins by peptide mapping after protein digestion by use of oriented immobilized proteinases.

Diagnostic methods for detecting gastric diseases using chymotryptic digestion of pepsin are discussed. Peptide maps can be prepared using reversed-phase high-performance liquid chromatography. Batchwise chromatography by use of membranes with immobilized Tyr(I2) was used for the isolation of pepsin from gastric mucosa extract or from human blood serum. Enzymes immobilized using suitable antibodies or through their sugar moieties can be used for the preparation of peptide maps because such enzymes share good steric accessibility to their active binding sites and possess increased thermal stability. Biospecific adsorption of proteins to immunosorbents combines the simultaneous isolation of these enzymes with their oriented immobilization. Proteins were stabilized by hydrophilization through the attachment of saccharide residues containing galactose residues. These residues could be activated by oxidation using galactose oxidase and subsequently immobilized to hydrazide-containing solid supports.

Diazotizable supports of potential interest as affinity chromatography matrices.

Immobilization of affinity ligands, proteins, enzymes and other functional groups by azo coupling is based on the high reactivity of the support-carrying diazonium groups towards both low- and high-molecular weight compounds containing certain groupings such as phenols, imidazole and some other heterocycles, thiols and amines. The precursor of diazonium group is a diazotizable amine on the matrix. Remarkable progress in its preparation was achieved by application of (4-amino-phenyl)-(2-sulphatoxyethyl) sulphone-type reagents for functionalization of the matrix. A similar type of amine precursor is prepared by the reaction of monosubstituted sulphanilamide with epoxide-containing matrix. The presence of a SO2 group in the para position to diazonium group of these supports (after diazotization) enhances reactivity in azo coupling. 'Reversed' azo coupling is the reaction of a matrix containing functional groups capable of reacting with diazonium groups of a ligand. Preparation of a suitable matrix and examples of diazotizable ligands are given.

Galactosylation as a tool for the stabilization and immobilization of proteins.

This paper presents a brief overview of the role that the carbohydrate moieties of biologically active glycoproteins play in the stabilization and oriented immobilization of these proteins on solid supports. The synthetic galactosylation of hydrophobic areas or their surroundings on the protein surface improves the structural stability of native proteins against inactivation by the interaction of water with hydrophobic clusters. The lowering of the degree solvation of tyrosine residues in galactosylated trypsin and the model substance N-carbobenzoxy-L-glutamyl-L-tyrosine was proved by Raman spectroscopy. D-Galactose residues can be selectively oxidized, either with periodate or enzymatically, and the aldehyde groups thus formed are used for the immobilization of glycoproteins on solid supports with hydrazide groups under mild conditions.

Carbohydrates as a tool for oriented immobilization of antigens and antibodies.

A biospecific sorbent for the isolation of ovalbumin antibodies was prepared by coupling of ovalbumin via its periodate-oxidized carbohydrate moiety to bead cellulose modified with adipic acid dihydrazide. The anti-ovalbumin IgG fraction isolated on this sorbent from immune rabbit serum contained only antibodies against protein determinants of ovalbumin. Thus, when these IgG were immobilized through their carbohydrate moieties to cellulose beads it became possible to prepare a biospecific sorbent for concanavalin A by oriented adsorption of ovalbumin. Ovalbumin was specifically adsorbed via its protein moiety and its carbohydrate part remained free for interaction with concanavalin A.

[Isolation of monoclonal antibodies from mouse ascitic fluid using chromatography on "blue" DEAE cellulose]. / Izolace monoklonálních protilátek z mysích ascitických tekutin chromatografií na "modré" DEAE celulóze.

A method is described of the isolation of monoclonal antibodies form mouse ascites fluids by chromatography on beaded DEAE cellulose with covalently bound Reactive Blue-2 ("Blue" DEAE cellulose). The method yields antibodies of 80-90% purity, free from protease activity.

Partial purification and characterization of anhydrotetracycline oxygenase of Streptomyces aureofaciens.

Anhydrotetracycline oxygenase was purified both by affinity chromatography and by hydrophobic interaction chromatography. Molecular weight of anhydrotetracycline oxygenase was determined to be 115,000 by Sephadex G-200 gel filtration. Using preparative isoelectric focusing the isoelectric point of the enzyme was estimated to be 5.3. The enzyme showed a sensitivity to thiol-specific inhibitors. During the hydrophobic interaction purification step, the activity dropped considerably. Reactivation occurred when a heat treated crude extract was added to the reaction mixture.