Centrifugal precipitation chromatography applied to fractionation of proteins with ammonium sulfate

A novel chromatographic system introduced here internally generates a concentration gradient of ammonium sulfate (AS) along a long channel to fractionate proteins according to their solubility in AS solution. The separation column consists of a pair of discs with mutually mirror-imaged spiral channels that are separated by a semipermeable membrane. The disc assembly is mounted on a seal-less continuous flow centrifuge. A concentrated AS solution is introduced into the upper channel while a water solution is passed through the lower channel in the opposite direction in a rotating column. A mixture of proteins injected into the water channel moves along an AS gradient of increasing concentration that has been established in the water solution. Each protein species precipitates at a different AS concentration along the gradient. By decreasing the concentration of the AS solution its concentration in the water is decreased, causing the protein to redissolve and to reprecipitate further down the channel. The eluate is continuously monitored and collected using a fraction collector. The method has been demonstrated on separation of serum proteins and applied to purification of a recombinant ketosteroid isomerase from a crude E. coli lysate by adding an affinity ligand to the sample solution. A possible mechanism involved in this affinity separation is discussed. The method may be applied to other biopolymers such as DNA and RNA.