One-step purification of recombinant yeast 6-phosphofructo-2-kinase after the identification of contaminants by MALDI-TOF MS

His-tagged yeast 6-phosphofructo-2-kinase was overexpressed in the yeast st rain DFY658 under the control of the Gall promoter. Here we describe a simp le and fast purification protocol for the recombinant enzyme under native c onditions using a HiTrap affinity column loaded with CuSO4. The use of MALD I-TOF MS after in-gel-digestion enabled us to identify a critical contamina tion of the end product as yeast alcohol dehydrogenase1 (Adh1p). After iden tification this contaminant could be efficiently removed by carrying out th e washing steps at 25 degreesC instead of at 4 degreesC. To reduce the cell ular proteolytic activities a low phosphate concentration in the growth med ium was applied. This simple modification of the yeast cell growth conditio ns increased significantly the yield of the recombinant protein. (C) 2001 A cademic Press.