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Production of pro‐opiomelanocortin (POMC) by a vaccinia virus transient expression system and in vitro processing of the expressed prohormone by POMC‐converting enzyme
Author(s) -
Andreasson K.I.,
Tam W.W.H.,
Feurst T.O.,
Moss B.,
Loh Y.P.
Publication year - 1989
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(89)80428-4
Subject(s) - prohormone , vaccinia , in vitro , enzyme , biology , chemistry , biochemistry , gene , recombinant dna , hormone
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 μg 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 β‐endorphin antisera. Labeling the transfected cells with [ 3 H]Arg, followed by immunoprecipitation and SDS‐PAGE showed the synthesis of a major peak of POMC, 33 kDa. Purified [ 3 H]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), β‐lipotropin and β‐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.