sepharose and 1-4-bis(2-3-epoxypropoxy)butane

At present, the common tool for affinity purification of IgG is immobilized Protein A, which is separated from native cell-wall components of Staphylococcus aureus. It is complicated and costly to prepare natural Protein A. ZZ protein is a synthetic Fc region-binding domain originated from B domain of Protein A. In the present study, recombinant ZZ protein with a hexahistidine tag at the N-terminus was expressed in BL21 (DE3) under the control of T7 promoter. The protein was purified through one-step Ni2+ chelating affinity chromatography at a yield of 50 mg of protein/litre of culture. Then it was covalently coupled with Sepharose 4B with butane-1,4-diol diglycidyl ether. The protein ZZ-Sepharose 4B resin exhibited good performance in affinity purification of IgG, as well as in capturing the protein-interacting complexes in immunoprecipitation experiments. Compared with natural Protein A, the expression and purification of ZZ protein at high yield are very simple and low-cost. At this point, extensive applications of protein ZZ in immunoassays are practicable and to be anticipated.

Topics: Butylene Glycols; Chromatography, Affinity; Cloning, Molecular; Cross-Linking Reagents; Histidine; Immunoglobulin G; Immunoprecipitation; Protein Structure, Tertiary; Recombinant Proteins; Sepharose; Staphylococcal Protein A

This study reevaluates the potential for direct coupling of NAD(P)(+) to a carboxylate-terminating spacer arm using carbodiimide-promoted coupling in an attempt to develop a greatly simplified synthetic method for cofactor immobilization that would support the more widespread adoption of kinetic-based enzyme capture (KBEC) strategies for protein purification applications and protein-detecting arrays/proteomic studies. Direct coupling of NAD(+) to epoxy (1,4-butanediol diglycidyl ether)-activated Sepharose is also described. Depending on the synthetic method used, the position of attachment of cofactor is concluded to be primarily through the pyrophosphate or ribosyl hydroxyl groups. Total substitution levels varied from 0.5 to 2 micromol/g wet weight with 28-67% accessibility. Model bioaffinity chromatographic studies employing KBEC strategies are reported for bovine heart L-lactate dehydrogenase, yeast alcohol dehydrogenase, l-phenylalanine dehydrogenase from Sporosarcina, glutamate dehydrogenase (GDH) from Candida utilis, and GDH from bovine liver. The NAD(+) derivative prepared using epoxy-activated Sepharose shows most potential for further development based on total substitution levels, the apparent absence of nonbiospecific interference, reversible biospecific adsorption of some of the test enzymes using soluble KBEC/stripping ligand tactics, and the relative simplicity of the synthetic method.

Topics: Adsorption; Alcohol Dehydrogenase; Animals; Bacillus; Butylene Glycols; Candida; Carbodiimides; Cattle; Enzymes, Immobilized; Glucosephosphate Dehydrogenase; Glutamate Dehydrogenase; Glutamate Dehydrogenase (NADP+); Kinetics; L-Lactate Dehydrogenase; Liver; NAD; NADP; Saccharomyces cerevisiae; Sepharose

This work presents studies on the interactions of supercoiled plasmid DNA and Escherichia coli genomic DNA (gDNA) and RNA, with an hydrophobic interaction chromatography (HIC) gel, obtained by derivatisation of Sepharose CL-6B with 1,4-butanediol diglycidyl ether. Nucleic acids purified from E. coli were injected separately in the above HIC column and eluted with 1.5 M (NH4)2SO4 in the buffer. The column was able to separate single-stranded from double-stranded nucleic acids. RNA and denatured gDNA were retarded in a different way due to the interactions of the exposed hydrophobic bases with the ligands. Supercoiled plasmid DNA, on the contrary, eluted in the flowthrough.

Topics: Butylene Glycols; Chromatography, Agarose; DNA, Bacterial; DNA, Superhelical; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Plasmids; RNA, Bacterial; Sepharose

During the use of chromatographic supports for the purification of proteins or the selective removal of substances by immunoaffinity, leakage of the antibodies immobilized on the matrix is systematically observed. When the cleansing of blood plasma by extracorporeal circulation is concerned, it is of prime importance that the immunoadsorbents exhibit an extensive chemical stability over the whole range of experimental conditions. To study and minimize this leakage, a matrix, Sepharose CL-4B, was activated by various chemical reagents and coupled to goat anti-apolipoprotein B polyclonal antibodies. Immunoadsorbents thus prepared were compared with those obtained earlier by cyanogen bromide activation. It turns out that divinyl sulphone- and tresyl chloride-activated supports lead to similar results in terms of coupling yield and adsorption capacity, but to a significant reduction in released antibodies.

Topics: Animals; Antibodies; Antibody Specificity; Apolipoproteins B; Butylene Glycols; Chromatography, Affinity; Cyanogen Bromide; Goats; Humans; Hydrogen-Ion Concentration; Immunosorbent Techniques; Immunosorbents; Sepharose; Sulfones

Macroporous agarose beads were converted into non-porous beads by shrinkage and cross-linking in organic solvents. These beads could be used for high-performance hydrophobic-interaction chromatography without derivatization with non-polar ligands, because the 1,4-butanediol diglycidyl ether, used as cross-linker, gives relatively hydrophobic bridges. The resolution for compressed columns packed with these beads was determined as a function of gradient time at constant flow-rate, flow-rate at constant gradient volume and flow-rate at constant gradient time and as a function of loading capacity. Interestingly, the resolution is virtually independent of flow-rate at constant gradient volume even when the column is packed with relatively large beads (diameter 30 microns). The beads have the advantage of being stable up to pH 14.

Topics: Butylene Glycols; Chemical Phenomena; Chemistry, Physical; Chromatography, High Pressure Liquid; Cross-Linking Reagents; Proteins; Sepharose

Topics: Butylene Glycols; Carbohydrates; Chromatography, Affinity; Epoxy Compounds; Glycoside Hydrolases; Hydrogen-Ion Concentration; Indicators and Reagents; Lectins; Ligands; Sepharose