화학공학소재연구정보센터
Biochemical and Biophysical Research Communications, Vol.371, No.4, 855-859, 2008
Expression of microsomal lanosterol 14 alpha-demethylase (CYP51) in an engineered soluble monomeric form
We prepared a soluble monomeric form of bovine cytochrome P450 lanosterol 14 alpha-demethylase (CYP51), which in mammals is a ubiquitously expressed membrane protein of the endoplasmic reticulum. We constructed two variants of bovine CYP51 (bCYP51) with different truncations and modifications in their N-terminal membrane-spanning domains. Both of these were expressed in Escherichia coli at levels of 500 nmol/l. The protein variants were purified and tested for the solubility in the absence of detergent. Variant bCYP51-d1 exhibited similar to 10-fold better solubility over variant bCYT51-d2. The bCYP51-d1 eluted as a single peak in size-exclusion chromatography, corresponding to its monomeric form. The activity of bCYP51-dl is similar to that of recombinant human CYP51 with a non-truncated membrane-spanning region. High solubility and low tendency to non-specific oligomer formation make bCYP51-d1 a promising candidate for successful crystallization, which may finally allow the structural determination of this important mammalian enzyme. (C) 2008 Elsevier Inc. All rights reserved.