Production of pro-opiomelanocortin (POMC) by a vaccinia virus transient expression system and in vitro processing of the expressed prohormone by POMC-converting enzyme.

Pro-opiomelanocortin (POMC) was expressed in CV-1 (green monkey kidney) cells using a vaccinia virus transient expression system [(1986) Proc. Natl. Acad. Sci. USA 83, 8122]. The system involved infection of cells with a recombinant vaccinia virus carrying the T7 RNA polymerase gene and transfection with a plasmid containing the mouse POMC sequence flanked by the T7 RNA polymerase promoter at its 5'-end and the T7 RNA polymerase terminator at its 3'-end. Assay of the medium from transfected cells showed that 1-2 micrograms of immunoreactive ACTH was produced/10(6) cells. Analysis of the same medium by SDS-PAGE/Western blots revealed a band of 30-36 kDa, which was immunostained with both ACTH and beta-endorphin antisera. Labeling the transfected cells with [3H]Arg, followed by immunoprecipitation and SDS-PAGE showed the synthesis of a major peak of POMC, 33 kDa. Purified [3H]POMC expressed by CV-1 cells was cleaved in vitro by bovine intermediate lobe secretory vesicle pro-opiomelanocortin-converting enzyme to ACTH intermediates (19-25 kDa), beta-lipotropin and beta-endorphin. Thus, this work has demonstrated a technique for expressing microgram quantities of prohormones in mammalian cells, suitable for use as substrates for prohormone-converting enzymes in vitro.