Expression of human P450c17 as an export protein in Saccharomyces cerevisiae.
Cytochrome P450c17 (P450c17), together with cytochrome P450c21 (P450c21), plays an important role in progesterone metabolism in the mammalian adrenal cortex. Low levels of expression and the presence of other steroidogenic enzymes in adrenal cortex endoplasmic reticulum (ER) impedes purification and characterisation of wild type as well as mutant forms of the hemoprotein. Heterologous gene expression systems have previously been used successfully to express active P450c17. Heterologous expression can also be used for the preparation of anti-P450c17-IgG. For antibody production larger amounts of pure P450c17 peptide, rather than the active protein, is, however, desirable. If the expressed protein can be affinity tagged and secreted into the medium, isolation and purification will be facilitated. Saccharomyces cerevisiae, YPH259, was transformed with a modified YCplac111 yeast expression-secretion vector (pPRL2). The gene coding for a truncated human P450c17 (signal anchor sequence 1-18 was removed) was inserted, in reading frame, downstream from the leader sequence MF alpha. A histidine tag was incorporated at the C-terminus. The modified yeast expression vector was expressed in yeast, the secreted P450c17-peptide purified by affinity chromatography and identified by immunoblot analysis.
Swart
A C
AC
Department of Biochemistry, University of Stellenbosch, South Africa.
Swart
P
P
Roux
S P
SP
van der Merwe
K J
KJ
Pretorius
I S
IS
Steyn
A J
AJ
D001692:Biological Transport; D003001:Cloning, Molecular; D004926:Escherichia coli; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae; D013254:Steroid 17-alpha-Hydroxylase / Q000096:biosynthesis / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013254:Steroid 17-alpha-Hydroxylase