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High-Level Expression of the Prohormones Proenkephalin, Pro-Neuropeptide Y, Proopiomelanocortin, and β-Protachykinin forin VitroProhormone Processing
- Source :
- Protein Expression and Purification. 10:80-88
- Publication Year :
- 1997
- Publisher :
- Elsevier BV, 1997.
-
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.
- Subjects :
- endocrine system
Proteases
DNA, Complementary
Pro-Opiomelanocortin
Genes, Viral
Swine
Recombinant Fusion Proteins
Blotting, Western
Prohormone
Prohormone convertase
Protein Sorting Signals
Proopiomelanocortin
Bacteriophage T7
Tachykinins
Endopeptidases
Escherichia coli
medicine
Animals
Neuropeptide Y
Cloning, Molecular
Protein Precursors
Promoter Regions, Genetic
Expression vector
biology
Enkephalins
Fusion protein
Rats
Proenkephalin
Biochemistry
biology.protein
Electrophoresis, Polyacrylamide Gel
Proprotein Convertases
Protein Processing, Post-Translational
Biotechnology
medicine.drug
Subjects
Details
- ISSN :
- 10465928
- Volume :
- 10
- Database :
- OpenAIRE
- Journal :
- Protein Expression and Purification
- Accession number :
- edsair.doi.dedup.....6f27cc76db1f8d880f9981c995f0d2e4