Affinity purification of lipid vesicles
| Autor: | Boris Peker, James R. Swartz, Jessica J. Wuu |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
Streptavidin
Biotin Chromatography Affinity Polyethylene Glycols chemistry.chemical_compound Membrane Lipids Affinity chromatography Particle Size Chromatography biology Vesicle Phosphatidylethanolamines Ligand (biochemistry) Avidin Binding constant chemistry Biotinylation Liposomes biology.protein Phosphatidylcholines lipids (amino acids peptides and proteins) Adsorption Biotechnology Protein Binding |
| Zdroj: | Biotechnology progress. 20(1) |
| ISSN: | 8756-7938 |
| Popis: | We present a novel column chromatography technique for recovery and purification of lipid vesicles, which can be extended to other macromolecular assemblies. This technique is based on reversible binding of biotinylated lipids to monomeric avidin. Unlike the very strong binding of biotin and biotin-functionalized molecules to streptavidin, the interaction between biotin-functionalized molecules and monomeric avidin can be disrupted effectively by ligand competition from free biotin. In this work, biotin-functionalized lipids (biotin-PEG-PE) were incorporated into synthetic lipid vesicles (DOPC), resulting in unilamellar biotinylated lipid vesicles. The vesicles were bound to immobilized monomeric avidin, washed extensively with buffer, and eluted with a buffer supplemented with free biotin. Increasing the biotinyl lipid molar ratio beyond 0.53% of all lipids did not increase the efficiency of vesicle recovery. A simple adsorption model suggests 1.1 x 10 13 active binding sites/mL of resin with an equilibrium binding constant of K = 1.0 x 10 8 M -1 . We also show that this method is very robust and reproducible and can accommodate vesicles of varying sizes with diverse contents. This method can be scaled up to larger columns and/or high throughput analysis, such as a 96-well plate format. |
| Databáze: | OpenAIRE |
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