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.
Andreasson
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KI
Section on Cellular Neurobiology, National Institute of Child Health & Human Development, Bethesda, MD 20892.
Tam
W W
WW
Feurst
T O
TO
Moss
B
B
Loh
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YP
D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D010450:Endopeptidases / Q000378:metabolism*; D000882:Haplorhini; D007668:Kidney / Q000378:metabolism; D010957:Plasmids; D011333:Pro-Opiomelanocortin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D043484:Proprotein Convertases; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014162:Transfection; D014616:Vaccinia virus / Q000235:genetics*; D001615:beta-Endorphin / Q000378:metabolism; D008083:beta-Lipotropin / Q000378:metabolism
D011994:Recombinant Proteins; D001615:beta-Endorphin; D011333:Pro-Opiomelanocortin; D000324:Adrenocorticotropic Hormone; D008083:beta-Lipotropin; D010450:Endopeptidases; D043484:Proprotein Convertases