CROSS-AXIS SYNCHRONOUS FLOW-THROUGH COIL PLANET CENTRIFUGE

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The type-XLL cross-axis synchronous flow-through coil planet centrifuge previously reported has been applied to separations of proteins using aqueous-aqueous polymer phase systems. The separation column consisted of a pair of multilayer coils each prepared from 2.6cm ID PTFE (polytetrafluorethylene) tubing by winding it onto a 3.8cm or 7.6cm 0D holder hub making 8 layers of left-handed coils. The total capacity of the column measured about 250ml for the smaller column and 280ml for the larger column. With a polymer phase system composed of polyethylene glycol 1000 and potassium phosphate each 12.5% (W/W) in distilled water, the smaller column produced higher retention of the stationary phase which exceeds 50% of the total column capacity at a flow rate of 1 ml/min. Using the smaller column, performance of the apparatus was evaluated in separations of stable protein mixture containing cytochrome C, myoglobin, ovalbumin and hemoglobin. All components were separated in 5 hours at partition efficiencies ranging from 550 to 35 theoretical plates, sharply decreasing in the order of elution. The method has been successfully applied to purification of recombinant enzymes such as uridine phosphorylase, purine nucleoside phosphorylase and reverse transcriptase. By optimizing the partition coefficient values, these enzymes were highly purified directly from crude E. coli homogenates in a single passage through the separation column.