His-tagged yeast 6-phosphofructo-2-kinase was overexpressed in the yeast strain DFY658 under the control of the Gall promoter. Here we describe a simple and fast purification protocol for the recombinant enzyme under native conditions using a HiTrap affinity column loaded with CuSO4. The use of MALDI-TOF MS after in-gel-digestion enabled us to identify a critical contamination of the end product as yeast alcohol dehydrogenase1 (Adh1p). After identification this contaminant could be efficiently removed by carrying out the washing steps at 25 degreesC instead of at 4 degreesC. To reduce the cellular proteolytic activities a low phosphate concentration in the growth medium was applied. This simple modification of the yeast cell growth conditions increased significantly the yield of the recombinant protein.