EXPRESSION OF HUMAN P450C17 AS AN EXPORT PROTEIN IN SACCHAROMYCES-CEREVISIAE

Cytochrome P450c17 (P450c17), together with cytochrome P450c21 (P450c2 1), plays an important role in progesterone metabolism in the mammalia n adrenal cortex. Low levels of expression and the presence of other s teroidogenic enzymes in adrenal cortex endoplasmic reticulum (ER) impe des purification and characterisation of wild type as well as mutant f orms of the hemoprotein. Heterologous gene expression systems have pre viously 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 pro tein can be affinity tagged and secreted into the medium, isolation an d purification will be facilitated. Saccharomyces cerevisiae, YPH259, was transformed with a modified YCplac111 yeast expression-secretion v ector (pPRL2). The gene coding for a truncated human P450c17 (signal a nchor sequence 1-18 was removed)was inserted, in reading frame, downst ream from the leader sequence MF alpha. A histidine tag was incorporat ed at the C-terminus. The modified yeast expression vector was express ed in yeast, the secreted P450c17-peptide purified by affinity chromat ography and identified by immunoblot analysis.