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

FEBS Letters

Loh, Y.P.²  Andreasson, K.I.²  Moss, B.¹  Feurst, T.O.¹  Tam, W.W.H.² 

[1] Laboratory of Viral Disease, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA;Section on Cellular Neurobiology, Laboratory of Neurochemistry and Neuroimmunology, National Institute of Child Health & Human Development, Bethesda, MD 20892, USA

关键词: Adrenocorticotropin/endorphin prohormone;    Prohormone processing;    Proopiomelanocortin-converting enzyme;    (CV-1 cell);    POMC;    pro-opiomelanocortin;    ACTH;    adrenocorticotropin;    α-MSH;    α-melanotropin or N α-acetyl-ACTH1–13 NH2;    LPH;    lipotropin;    β-END;    β-endorphin;    γ-MSH;    γ-melanotropin;    SDS-PAGE;    SDS-polyacrylamide gel electrophoresis;   

DOI  :  10.1016/0014-5793(89)80428-4

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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/10⁶ 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 [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), 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.

【 授权许可】

Unknown