Purification and characterization of bovine steroid 21-hydroxylase (P450c21) efficiently expressed in Escherichia coli.
Steroid 21-hydroxylase, P450c21, is responsible for the conversion of progesterone and 17alpha-hydroxyprogesterone to their 21-hydroxylated derivatives. P450c21 has been poorly investigated because of difficulty in obtaining sufficient quantities of purified protein. To solve the problem, we have attempted to express the bovine P450c21 in Escherichia coli as a stable form. The N-terminal membrane anchor and basic regions of P450c21 were replaced by the basic region of CYP2C3. The engineered P450c21 was expressed at a level higher than 1.2micromol/L culture (>60mg/L) when coexpressed with molecular chaperones GroES/GroEL. Utilizing three steps of column chromatography, the protein was highly purified to the specific content 16.6nmol/mg (91.2% purity). The purified protein is a monomer in the presence of 1% sodium cholate as determined by gel filtration analysis, suggesting that this membrane anchor-truncated form of P450c21 is more soluble than the native form. The purified enzyme showed typical substrate-binding difference spectra and 21-hydroxylase activities for both progesterone and 17alpha-hydroxyprogesterone. Truncation of the membrane anchor increases solubility of P450c21 facilitating expression of this protein in E. coli yielding sufficient quantities for both biochemical and biophysical studies.
Arase
Miharu
M
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
Waterman
Michael R
MR
Kagawa
Norio
N
D019326:17-alpha-Hydroxyprogesterone / Q000737:chemistry; D000818:Animals; D002417:Cattle / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D011374:Progesterone / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D012995:Solubility; D013255:Steroid 21-Hydroxylase / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification
D011994:Recombinant Proteins; C400322:cytochrome P-450 CYP2C subfamily; D011374:Progesterone; D019326:17-alpha-Hydroxyprogesterone; D003577:Cytochrome P-450 Enzyme System; D013255:Steroid 21-Hydroxylase