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  • Title: One-step purification of recombinant yeast 6-phosphofructo-2-kinase after the identification of contaminants by MALDI-TOF MS.
    Author: Dihazi H, Kessler R, Eschrich K.
    Journal: Protein Expr Purif; 2001 Feb; 21(1):201-9. PubMed ID: 11162407.
    Abstract:
    His-tagged yeast 6-phosphofructo-2-kinase was overexpressed in the yeast strain DFY658 under the control of the Gal1 promoter. Here we describe a simple and fast purification protocol for the recombinant enzyme under native conditions using a HiTrap affinity column loaded with CuSO(4). 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 degrees C instead of at 4 degrees C. 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.
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