Rapid purification yielding highly active 17 beta-hydroxysteroid dehydrogenase: application of hydrophobic interaction and affinity fast protein liquid chromatography.
Homogeneous human placental 17 beta-hydroxysteroid dehydrogenase was obtained by a procedure consisting of two fast protein liquid chromatographic (FPLC) steps using Phenyl-Sepharose hydrophobic interaction and Blue-Sepharose affinity columns. In the first chromatography, the enzyme eluted only when an additional decrease in ionic strength was inserted after the ammonium sulphate concentration had reached zero, thus enhancing the separation. In the affinity chromatography, separation of contaminating proteins occurred at different stages of loading and washing. The specific elution of the enzyme by the co-factor NADP+ is very efficient in obtaining a homogeneous preparation in high yield. The rapidity of FPLC was further increased by a maximum simplification of the intermediate steps, and the whole procedure lasted only two days. This preparation has a yield of more than 50% and a high specific activity, catalysing the formation of 7.9 mumol of estrone from estradiol per minute at pH 9.2 and 23 degrees C. It has an apparent molecular mass of 35,000. This provides an efficient candidate for the purification of other membrane-associated proteins.
Yang
F
F
MRC Group in Molecular Endocrinology, CHUL Research Center, Ste-Foy, Quebec, Canada.
Zhu
D W
DW
Wang
J Y
JY
Lin
S X
SX
D015067:17-Hydroxysteroid Dehydrogenases / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D009249:NADP; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy
D009249:NADP; D015067:17-Hydroxysteroid Dehydrogenases