Affinity chromatography using biocompatible and reusable biotinylated membranes.
A novel, reusable biotinylated affinity chromatography strategy for the bio-specific binding of bioactive avidin tagged enzymes or polypeptides is reported. Using an avidin coupled peroxidase fusion protein as a test system; non-specific protein shielding and matrix regeneration were also shown. The amphiphilic surfactant Pluronic F108 was used as an affinity linker, by non-covalent binding to membrane chromatographic matrices while the terminal hydroxyl groups of Pluronic were covalently coupled to the biological ligand biotin. Planar nonporous membranes of varying surface chemistry were synthesised to test the matrix dependent affinity binding of biotinylated Pluronic and their respective ability to resist non-specific protein adsorption. Membrane regeneration using sodium dodecyl sulphate (SDS) was capable of displacing both adsorbed proteins and Pluronic. SDS micelles (34 mM) were effective in desorbing membrane bound protein while 5mM SDS removed up to 85% of the bound ligand after 20 h incubation at 20 degrees C. In this study, polyvinylidene membranes had the highest ligand binding capacity of 0.22 mg cm(-2) and specific, competitive affinity binding of avidin-peroxidase was shown in the presence of up to 0.2 mg ml(-1) 'contaminant' proteins. The resultant biocompatible affinity chromatographic system was regenerated and reused with no significant change in performance for up to five cycles.
Govender
S
S
Department of Biochemistry, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa.
Jacobs
E P
EP
Bredenkamp
M W
MW
Swart
P
P
D000327:Adsorption; D001360:Avidin / Q000737:chemistry / Q000378:metabolism; D001710:Biotin / Q000737:chemistry*; D019921:Biotinylation; D002846:Chromatography, Affinity / Q000379:methods*; D008024:Ligands; D008567:Membranes, Artificial; D008954:Models, Biological; D009195:Peroxidase / Q000737:chemistry / Q000378:metabolism; D011145:Polyvinyls / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005465:Fluorocarbon Polymers
D001360:Avidin; D001710:Biotin; D008024:Ligands; D008567:Membranes, Artificial; D009195:Peroxidase; D011145:Polyvinyls; D011993:Recombinant Fusion Proteins; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers