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 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.
Dihazi
H
H
Medical Faculty, Institute of Biochemistry, University of Leipzig, Liebigstrasse 16, D-04103 Leipzig, Germany.
Kessler
R
R
Eschrich
K
K
D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D019327:Copper Sulfate; D017931:DNA Primers; D004340:Drug Contamination; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D025481:Phosphofructokinase-2; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D017931:DNA Primers; D011994:Recombinant Proteins; D000426:Alcohol Dehydrogenase; D017853:Phosphotransferases (Alcohol Group Acceptor); D025481:Phosphofructokinase-2; D019327:Copper Sulfate