Rapid purification of calmodulin and S-100 protein by affinity chromatography with melittin immobilized to Sepharose

Abstract

Melittin-Sepharose was prepared for Ca 2+-dependent affinity chromatography of calmodulin and S-100 protein. This matrix exhibits extremely high capacity (∼ 10 mg calmodulin/ml gel), low nonspecific binding, and excellent recovery (> 90%) under optimal conditions. Recovery of calmodulin from melittin-Sepharose was related to the degree of saturation of column capacity with lower yields when only partial saturation was achieved. Large-scale, simultaneous purification of calmodulin and S-100 protein from brain was carried out using selective adsorption to organomercurial agarose followed by melittin-Sepharose chromatography; yields were 250–300 mg of calmodulin and 200–300 mg of S-100 per kg tissue. Calmodulin also was purified in a single step from bovine testis supernatant using melittin-Sepharose in yields comparable to those from brain.