High-level expression of the prohormones proenkephalin, pro-neuropeptide Y, proopiomelanocortin, and beta-protachykinin for in vitro prohormone processing.
Protein Expression and Purification
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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.
Animals; Bacteriophage T7/genetics; Blotting; Western; Cloning; Cloning; Molecular; DNA; DNA; Complementary/genetics; Electrophoresis; Electrophoresis; Polyacrylamide Gel; Endopeptidases; Endopeptidases/metabolism; Enkephalins; Enkephalins/genetics; Enkephalins/metabolism; Escherichia coli; Genes; Genes; Viral; Immunoblotting; Molecular cloning; Neuropeptide Y; Neuropeptide Y/genetics; Neuropeptide Y/metabolism; Neurotransmitters; Polyacrylamide gel electrophoresis; Proopiomelanocortin; Pro-Opiomelanocortin/genetics; Pro-Opiomelanocortin/metabolism; Promoter Regions; Genetic; Proprotein convertases; Protein precursors; Protein Precursors/genetics; Protein Precursors/metabolism; Protein Processing; Post-Translational; Protein Sorting Signals/metabolism; Rats; Recombinant Fusion Proteins/metabolism; Swine; Tachykinins; Tachykinins/genetics; Tachykinins/metabolism; Western immunoblotting
Biochemistry | Genetics and Genomics | Molecular Biology | Molecular Genetics
Hook, V. Y.,
Lively, M. O.,
Schiller, M. R.,
High-level expression of the prohormones proenkephalin, pro-neuropeptide Y, proopiomelanocortin, and beta-protachykinin for in vitro prohormone processing..
Protein Expression and Purification, 10(1),