Calcium-modulated conformational affinity chromatography. Application to the purification of calmodulin and S100 proteins.
The purification of proteins by affinity chromatography is based on their highly specific interaction with an immobilized ligand followed by elution under conditions where their affinity towards the ligand is markedly reduced. Thus, a high-degree purification by a single chromatographic step is achieved. However, when several proteins in the crude mixture share affinity to a common immobilized ligand, they may not be resolved by affinity chromatography and subsequent "real" chromatographic purification steps may be required. It is shown that by using properly selected gradient elution conditions, the affinities of the various proteins towards the immobilized ligand may be gradually modulated and their separation may be achieved. This is exemplified by the isolation and separation of a group of Ca(2+)-activated proteins, Calmodulin, S100a and S100b, from bovine brain extract, using a melittin-Eupergit C affinity column which is developed with Ca(2+)-chelator gradients. As expected, separation of the three proteins into individual peaks, eluted in order of increasing affinity to the matrix, was obtained. Sigmoid selectivity curves calculated from the elution volumes under different elution conditions for each of the proteins were obtained, illustrating the chromatographic behaviour of the gradient affinity separation system.
Fleminger
G
G
Department of Molecular Microbiology and Biotechnology, George Wise Faculty of Life Sciences, Tel Aviv University, Israel.
Neufeld
T
T
Star-Weinstock
M
M
Litvak
M
M
Solomon
B
B
D000818:Animals; D002118:Calcium / Q000737:chemistry*; D002147:Calmodulin / Q000302:isolation & purification*; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D007457:Iodine Radioisotopes; D008555:Melitten / Q000737:chemistry; D008968:Molecular Conformation; D011108:Polymers; D009418:S100 Proteins / Q000302:isolation & purification*; D014020:Tissue Extracts / Q000032:analysis
D002147:Calmodulin; D007457:Iodine Radioisotopes; D011108:Polymers; D009418:S100 Proteins; D014020:Tissue Extracts; D008555:Melitten; C065843:Eupergit C; D002118:Calcium