Document Detail


Production of pro-opiomelanocortin (POMC) by a vaccinia virus transient expression system and in vitro processing of the expressed prohormone by POMC-converting enzyme.
MedLine Citation:
PMID:  2542089     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
K I Andreasson; W W Tam; T O Feurst; B Moss; Y P Loh
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  FEBS letters     Volume:  248     ISSN:  0014-5793     ISO Abbreviation:  FEBS Lett.     Publication Date:  1989 May 
Date Detail:
Created Date:  1989-07-05     Completed Date:  1989-07-05     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0155157     Medline TA:  FEBS Lett     Country:  NETHERLANDS    
Other Details:
Languages:  eng     Pagination:  43-7     Citation Subset:  IM    
Affiliation:
Section on Cellular Neurobiology, National Institute of Child Health & Human Development, Bethesda, MD 20892.
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MeSH Terms
Descriptor/Qualifier:
Adrenocorticotropic Hormone / metabolism
Animals
Cell Line
Endopeptidases / metabolism*
Haplorhini
Kidney / metabolism
Plasmids
Pro-Opiomelanocortin / biosynthesis*,  genetics,  isolation & purification
Proprotein Convertases
Recombinant Proteins / biosynthesis*
Transfection
Vaccinia virus / genetics*
beta-Endorphin / metabolism
beta-Lipotropin / metabolism
Chemical
Reg. No./Substance:
0/Recombinant Proteins; 60617-12-1/beta-Endorphin; 66796-54-1/Pro-Opiomelanocortin; 9002-60-2/Adrenocorticotropic Hormone; 9035-55-6/beta-Lipotropin; EC 3.4.-/Endopeptidases; EC 3.4.-/Proprotein Convertases

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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