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Affinity Support (affinity + support)

Distribution by Scientific Domains
Life Sciences75%

Selected Abstracts

A Novel Magnetic Affinity Support for Protein Adsorption and Purification

BIOTECHNOLOGY PROGRESS, Issue 1 2001
Xiao-Dong Tong
A novel magnetic support was prepared by an oxidization-precipitation method with poly(vinyl alcohol) (PVA) as the entrapment material. Transmission electron microscopy indicated that the magnetic particles had a core-shell structure, containing many nanometer-sized magnetic cores stabilized by the cross-linked PVA. The particles showed a high magnetic responsiveness in magnetic field, and no aggregation of the particles was observed after the particles had been treated in the magnetic field. These facts indicated that the particles were superparamagnetic. Cibacron blue 3GA (CB) was coupled to the particles to prepare a magnetic affinity support (MAS) for protein adsorption. Lysozyme was used as a model protein to test the adsorption properties of the MAS. The adsorption equilibrium of lysozyme to the MAS was described by the Langmuir-type isotherm. The capacity for lysozyme adsorption was more than 70 mg/g MAS (wet weight) at a relatively low CB coupling density (3,5 ,mol/g). In addition, 1.0 M NaCl solution could be used to dissociate the adsorbed lysozyme. Finally, the MAS was recycled for the purification of alcohol dehydrogenase (ADH) from clarified yeast homogenates. Under proper conditions, the magnetic separation yielded over 5-fold purification of the enzyme with 60% recovery of the enzyme activity. [source]

PURIFICATION OF AMYLASE FROM TILAPIA BY MAGNETIC PARTICLE

JOURNAL OF FOOD PROCESSING AND PRESERVATION, Issue 1 2010
MING CHANG WU
ABSTRACT Recent development in magnetic carrier technology involves the use of nonmagnetic substrates attached to superparamagnetic particles forming functionally modified magnetic support to isolate a particular enzyme in a controllable magnetic field. In this study, the superparamagnetic particles were modified by epichlorohydrin and other agents to cross-link with starch to form the purification support. This affinity support was able to bind the amylase and was used in the purification of amylase from Taiwan tilapia. After ammonium sulfate precipitation of amylase from Taiwan tilapia, the modified superparamagnetic particles were able to purify the crude amylase by 20.78-fold with recovery of activity of 75.6%. The molecular weight of the amylase was estimated to be 66.1 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both crude and purified amylase reached an optimum at a pH of 8.0 and temperature of 50C, and the enzyme was stable between 20 and 50C. PRACTICAL APPLICATIONS Because of the rapid development of high technology such as carrier supports for enzyme purification, the development, research and application of magnetic carriers are timely needed. The present study demonstrated that the affinity superparamagnetic particles could be used as a carrier support to absorb and purify the amylase and that technology of affinity purification can be widely used in protein purification. Compared with the traditional chromatography used in the purification of proteins, this novel affinity superparamagnetic particle technology is rapid, has low operation cost, requires simple facilities, and involves easy separation and recovery of the enzymes. [source]

A Novel Magnetic Affinity Support for Protein Adsorption and Purification

BIOTECHNOLOGY PROGRESS, Issue 1 2001
Xiao-Dong Tong
A novel magnetic support was prepared by an oxidization-precipitation method with poly(vinyl alcohol) (PVA) as the entrapment material. Transmission electron microscopy indicated that the magnetic particles had a core-shell structure, containing many nanometer-sized magnetic cores stabilized by the cross-linked PVA. The particles showed a high magnetic responsiveness in magnetic field, and no aggregation of the particles was observed after the particles had been treated in the magnetic field. These facts indicated that the particles were superparamagnetic. Cibacron blue 3GA (CB) was coupled to the particles to prepare a magnetic affinity support (MAS) for protein adsorption. Lysozyme was used as a model protein to test the adsorption properties of the MAS. The adsorption equilibrium of lysozyme to the MAS was described by the Langmuir-type isotherm. The capacity for lysozyme adsorption was more than 70 mg/g MAS (wet weight) at a relatively low CB coupling density (3,5 ,mol/g). In addition, 1.0 M NaCl solution could be used to dissociate the adsorbed lysozyme. Finally, the MAS was recycled for the purification of alcohol dehydrogenase (ADH) from clarified yeast homogenates. Under proper conditions, the magnetic separation yielded over 5-fold purification of the enzyme with 60% recovery of the enzyme activity. [source]

A novel purification strategy for retrovirus gene therapy vectors using heparin affinity chromatography

BIOTECHNOLOGY & BIOENGINEERING, Issue 4 2005
Marķa de las Mercedes Segura
Abstract Membrane separation and chromatographic technologies are regarded as an attractive alternative to conventional academic small-scale ultracentrifugation procedures used for retrovirus purification. However, despite the increasing demands for purified retroviral vector preparations, new chromatography adsorbents with high specificity for the virus have not been reported. Heparin affinity chromatography is presented here as a novel convenient tool for retrovirus purification. The ability of bioactive retroviral particles to specifically bind to heparin ligands immobilized on a chromatographic gel is shown. A purification factor of 63 with a recovery of 61% of functional retroparticles was achieved using this single step. Tentacle heparin affinity supports captured retroviral particles more efficiently than conventional heparin affinity chromatography supports with which a lower recovery was obtained (18%). Intact, infective retroviral particles were recovered by elution with low salt concentrations (350 mM NaCl). Mild conditions for retrovirus elution from chromatographic columns are required to preserve virus infectivity. VSV-G pseudotyped retroviruses have shown to be very sensitive to high ionic strength, losing 50% of their activity and showing membrane damage after a short exposure to 1M NaCl. We also report a complete scaleable downstream processing scheme for the purification of MoMLV-derived vectors that involves sequential microfiltration and ultra/diafiltration steps for virus clarification and concentration respectively, followed by fractionation by heparin affinity chromatography and final polishing by size-exclusion chromatography. Overall, by using this strategy, a 38% yield of infective particles can be achieved with a final purification factor of 2,000. © 2005 Wiley Periodicals, Inc. [source]