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| - Selection of Ligands for Affinity ChromatographyUsing Quartz Crystal Biosensor
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| - This paper described a new strategy for rapid selectingligands for application in affinity chromatography using aquartz crystal microbalance (QCM) biosensor. An aminoglycoside antibiotic drug, kanamycin (KM), was immobilized on the gold electrodes of the QCM sensor chip.The binding interactions of the immobilized KM withvarious proteins in solution were monitored as the variations of the resonant frequency of the modified sensor.Such a rapid screen analysis of interactions indicatedclearly that KM-immobilized sensor showed strong specific interaction only with lysozyme (LZM). The resultantsensorgrams were rapidly analyzed by using a kineticanalysis software based on a genetic algorithm to deriveboth the kinetic rate constants (kass and kdiss) and equilibrium dissociation constants (KD) for LZM−KM interactions. The immobilized KM showed higher affinity to LZMwith a dissociation constant on the order of 10-5 M, whichis within the range of 10-4−10-8 M and suitable for anaffinity ligand. Therefore, KM was demonstrated for thefirst time as a novel affinity ligand for purification of LZMand immobilized onto the epoxy-activated silica in thepresence of a high potassium phosphate concentration.The KM immobilized affinity column has proved usefulfor a very convenient purification of LZM from chickenegg white. The purity of LZM obtained was higher than90%, as determined by densitometric scanninng of sodium dodecyl sulfate-polyacrylamide gel electrophoresisof purified fraction. These results confirmed that theselected KM ligand is indeed a valuable affinity ligand forpurification of LZM. The new screening strategy based ona QCM biosensor is expected to be a promising way forrapid selecting specific ligands for purifying other valuableproteins.
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