High-level expression of the prohormones proenkephalin, pro-neuropeptide Y, proopiomelanocortin, and beta-protachykinin for in vitro prohormone processing.
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.
Hook
V Y
VY
Department of Medicine, University of California, San Diego, La Jolla 92093-0822, USA. vhook@ucsd.edu
Moran
K
K
Kannan
R
R
Kohn
A
A
Lively
M O
MO
Azaryan
A
A
Schiller
M
M
Miller
K
K
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
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