title: High-level expression of the prohormones proenkephalin, pro-neuropeptide Y, proopiomelanocortin, and beta-protachykinin for in vitro prohormone processing.
abstract: Prohormone substrates are required for investigation of the proteolytic processing of prohormones and proproteins into active peptide hormones and neurotransmitters. However, the lack of prohormone proteins has been a limiting factor in elucidating proteolytic mechanisms for conversion of prohormones into active peptides. Therefore, in this study, cloned cDNAs encoding the prohormones proenkephalin (PE), pro-neuropeptide Y (pro-NPY), pro-opiomelanocortin (POMC), and beta-protachykinin (beta-PT) were utilized to express recombinant prohormones in Escherichia coli. High-level expression of milligrams of prohormones was achieved with the pET3c expression vector utilizing the T7 promoter for production of PE, pro-NPY, and POMC, as demonstrated by SDS-PAGE gel electrophoresis, Western blots, and 35S-methionine labeling. In addition, beta-PT was expressed at high levels as fusion proteins with the maltose-binding protein and glutathione S-transferase by the pMAL-c and pGEX-2T expression vectors, respectively. Relative rates of processing by the established processing proteases "prohormone thiol protease" (PTP), 70-kDa aspartyl protease, and PC1/ 3 and PC2 (PC, prohormone convertase) were examined with purified PE, pro-NPY, and POMC. Distinct preferences of processing enzymes for different prohormones was demonstrated. PTP preferred PE and pro-NPY substrates, whereas little processing of POMC was detected. In contrast, the 70-kDa aspartyl protease cleaved POMC more readily than pro-NPY or PE. However, PC1/3 and PC2 prefer POMC as substrate. Demonstration of selectivity of processing enzymes for prohormone substrates illustrates the importance of expressing recombinant prohormones for in vitro processing studies.
authors.affiliation: Department of Medicine, University of California, San Diego, La Jolla 92093-0822, USA. vhook@ucsd.edu
authors.forename: V Y
authors.initials: VY
authors.lastname: Hook
authors.forename: K
authors.initials: K
authors.lastname: Moran
authors.forename: R
authors.initials: R
authors.lastname: Kannan
authors.forename: A
authors.initials: A
authors.lastname: Kohn
authors.forename: M O
authors.initials: MO
authors.lastname: Lively
authors.forename: A
authors.initials: A
authors.lastname: Azaryan
authors.forename: M
authors.initials: M
authors.lastname: Schiller
authors.forename: K
authors.initials: K
authors.lastname: Miller
mesh_terms: D000818:Animals; D017123:Bacteriophage T7 / Q000235:genetics; D015153:Blotting, Western; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D004745:Enkephalins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli; D005814:Genes, Viral; D009478:Neuropeptide Y / Q000235:genetics* / Q000378:metabolism; D011333:Pro-Opiomelanocortin / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013552:Swine; D015320:Tachykinins / Q000235:genetics* / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D004745:Enkephalins; D009478:Neuropeptide Y; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D015320:Tachykinins; C029992:proenkephalin; C054618:protachykinin; C081158:proneuropeptide Y; D011333:Pro-Opiomelanocortin; D010450:Endopeptidases