sepharose and betadex

In this article, we propose an impressive and facile strategy to improve protein refolding using solid phase artificial molecular chaperones consisting of the surface-functionalized magnetic nanoparticles. Specifically, monotosyl-β-cyclodextrin connected to the surface of 3-aminopropyltriethoxysilane (APES)-modified magnetic nanoparticles is immobilized on the sepharose surface to promote interaction with exposed hydrophobic surfaces of partially folded (intermediates) and unfolded states of proteins. Their efficiencies were investigated by circular dichroism spectroscopy and photoluminescence spectroscopy of the protein. Although the mechanism of this method is based on principles of hydrophobic chromatography, this system is not only purging the native protein from inactive inclusion bodies but also improving the protein refolding process. We chose β-cyclodextrin (β-CD) considering multiple reports in the literature about its efficiency in protein refolding and its biocompatibility. To increase the surface area/volume ratio of the sepharose surface by nanoparticles, more β-CD molecules are connected to the sepharose surface to make a better interaction with proteins. We suppose that proteins are isolated in the nanospace created by bound cyclodextrins on the resin surface so intermolecular interactions are reduced. The architecture of nanoparticles was characterized by Fourier transform infrared spectra, X-ray diffraction, scanning electron microscopy images, energy dispersive X-ray spectroscopy, nuclear magnetic resonance (

Topics: alpha-Amylases; beta-Cyclodextrins; Ferrosoferric Oxide; Magnetite Nanoparticles; Nuclear Magnetic Resonance, Biomolecular; Propylamines; Protein Refolding; Proteins; Sepharose; Silanes; Spectrometry, X-Ray Emission; Spectroscopy, Fourier Transform Infrared

An oligomerized beta-cyclodextrin ligand coupled to brominated allyl-group substituted Sepharose HP has been used for the one-step purification of polyphenolic epigallocatechin gallate (EGCG), an important antioxidant, by isocratic hydrogen bond adsorption chromatography. With a sample load of 1.33 mg crude green tea polyphenolic extract per ml column packing and with water/ethanol/acetonitrile (57/30/13, v/v) as the optimum mobile phase, an EGCG purity of about 98% with a recovery of approximate 73% could be achieved by proper peak cutting. After about 10 sample applications, the column performance started to deteriorate but could be regenerated to its original function by cleaning with 0.35 M NaOH.

Topics: beta-Cyclodextrins; Catechin; Chromatography, High Pressure Liquid; Hydrogen Bonding; Mass Spectrometry; Plant Extracts; Sepharose; Tea

The isoflavonoid puerarin, a well-known traditional Chinese drug, has been purified in one step from an extract of Radix puerariae (root of the plant Pueralria lobata) by adsorption chromatography on an epichlorohydrin polymerized beta-cyclodextrin ligand coupled to brominated allyl-group substituted Sepharose HP. Acetic acid (10%) was used as the mobile phase and the optimum loading capacity was around 1.2 mg crude extract/ml packed gel. The purity of the collected puerarin was about 98% with a recovery of about 62%.

Topics: beta-Cyclodextrins; Chromatography, High Pressure Liquid; Cyclodextrins; Isoflavones; Sepharose

Two alpha-amylase isoforms designated VAAmy1 and VAAmy2 were purified from cotyledons of germinating seedlings of azuki bean (Vigna angularis). VAAmy1 apparently had lower affinity towards a beta-cyclodextrin Sepharose column than VAAmy2. Molecular weights of VAAmy1 and VAAmy2 were estimated to be 47,000 and 44,000, respectively. However, no considerable difference was found between them in effects of pH, temperature, CaCl2, and EDTA, as well as the kinetic parameters for amylose (average degree of polymerization 17): kcat, 71.8 and 55.5 s(-1), Km, 0.113 and 0.097 mg/ml; for blocked 4-nitrophenyl alpha-D-maltoheptaoside: kcat, 62.4 and 85.3 s(-1), Km, 0.22 and 0.37 mM, respectively. Primary structures of the two enzymes were analyzed by N-terminal sequencing, cDNA cloning, and MALDI-TOF mass spectrometry, implying that the two enzymes have the same peptide. The results indicated that the low affinity of VAAmy1 towards beta-cyclodextrin Sepharose was due to some modification on/near carbohydrate binding site in the limited sequence regions, resulting in higher molecular weight.

Topics: alpha-Amylases; Amino Acid Sequence; beta-Cyclodextrins; Calcium; Chelating Agents; Chromatography, Affinity; Cyclodextrins; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Fabaceae; Hydrogen-Ion Concentration; Hydrolysis; Isoenzymes; Kinetics; Molecular Sequence Data; Plant Extracts; Protein Processing, Post-Translational; Reverse Transcriptase Polymerase Chain Reaction; Sepharose; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Starch; Substrate Specificity; Temperature; Trypsin