Generation of catalytically active 6-phosphofructokinase from Saccharomyces cerevisiae in a cell-free system

PFK1 and PFK2 coding for the subunits of 6-phosphofructokinase from Sacchar omyces cerevisiae were cloned into plasmids suitable for runoff transcripti on. In vitro translation products of both kinds of subunit were obtained us ing rabbit reticulocyte lysate as the synthesis and folding system. They we re monitored by chemiluminescent Western-blot analysis. Folding and assembl y of the alpha-subunit and beta-subunit of 6-phosphofructokinase were found to occur in the cell-free system resulting in an enzymatically active prot ein. The in vitro generated enzyme exhibits a folding state that is similar to that of the heterooctameric form of 6-phosphofructokinase in the presen ce of fructose 6-phosphate, ATP and ammonium sulfate, as demonstrated by si ze-exclusion HPLC followed by ELISA.