PURIFICATION BY REFLUX ELECTROPHORESIS OF WHEY PROTEINS AND OF A RECOMBINANT PROTEIN EXPRESSED IN DICTYOSTELIUM-DISCOIDEUM

Protein purification that combines the use of molecular mass exclusion membranes with electrophoresis is particularly powerful as it uses pr operties inherent to both techniques. The use of membranes allows effi cient processing and is easily scaled up, while electrophoresis permit s high resolution separation under mild conditions. The Gradiflow appa ratus combines these two technologies as it uses polyacrylamide membra nes to influence electrokinetic separations. The reflux electrophoresi s process consists of a series of cycles incorporating a forward phase and a reverse phase. The forward phase involves collection of a targe t protein that passes through a separation membrane before trailing pr oteins in the same solution. The forward phase is repeated following c learance of the membrane in the reverse phase by reversing the current . We have devised a strategy to establish optimal reflux separation pa rameters, where membranes are chosen for a particular operating range and protein transfer is monitored at different pH values. In addition, forward and reverse phase times are determined during this process. T wo examples of the reflux method are described. In the first case, we describe the purification strategy for proteins from a complex mixture which contains proteins of higher electrophoretic mobility than the t arget protein. This is a two-step procedure, where first proteins of h igher mobility than the target protein are removed from the solution b y a series of reflux cycles, so that the target protein remains as the leading fraction. In the second step the target protein is collected, as it has become the leading fraction of the remaining proteins. In t he second example we report the development of a reflux strategy which allowed a rapid one-step preparative purification of a recombinant pr otein, expressed in Dictyostelium discoideum. These strategies demonst rate that the Gradiflow is amenable to a wide range of applications, a s the protein of interest is not necessarily required to be the leadin g fraction in solution. (C) 1997 Elsevier Science B.V.