Centrifugal precipitation chromatography: principle, apparatus, and optimization of key parameters for protein fractionation by ammonium sulfate precipitation.
A novel chromatographic system introduced here internally generates a concentration gradient of ammonium sulfate (AS) through a long separation channel under a centrifugal force field. Protein samples are exposed to a gradually increasing AS concentration and precipitated along the channel. Then, chromatographic elution is initiated by gradually decreasing the AS concentration in the gradient which causes the proteins to repeat dissolution and precipitation through the channel. Consequently, they are eluted out in the order of their solubility in the AS solution. The separation column consists of a pair of disks equipped with mutually mirror-imaged spiral grooves. A dialysis membrane is sandwiched between the disks to form two identical channels partitioned by the membrane. The disk assembly is mounted on the sealless continuous-flow centrifuge. When a concentrated AS solution is eluted through one channel and water through the other channel in an opposite direction, an exponential AS gradient is formed through the water channel. A series of basic experiments was performed to study the rates of AS transfer and osmosis through the membrane, and the operational parameters including elution time, revolution speed, inclination of gradient, and sample size were optimized using stable protein samples. Preliminary applications were successful in purification of monoclonal antibody from cell culture supernatant and an affinity separation of recombinant ketosteroid isomerase from a crude Escherichia coli lysate.
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Laboratory of Biophysical Chemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 10, Room 7N322, Bethesda, Maryland, 20892-1676, USA.
D000645:Ammonium Sulfate; D002021:Buffers; D002498:Centrifugation / Q000295:instrumentation / Q000379:methods; D011232:Chemical Precipitation; D003956:Dialysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D011506:Proteins / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification; D012709:Serum Albumin / Q000302:isolation & purification*; D013251:Steroid Isomerases / Q000302:isolation & purification; D005719:gamma-Globulins / Q000302:isolation & purification*
D002021:Buffers; D011506:Proteins; D011994:Recombinant Proteins; D012709:Serum Albumin; D005719:gamma-Globulins; D013251:Steroid Isomerases; C022660:steroid delta-isomerase; D000645:Ammonium Sulfate