High-level expression of the prohormones proenkephalin, pro-neuropeptide Y, proopiomelanocortin, and beta-protachykinin for in vitro prohormone processing

Protein Expression and Purification
V Y HookK Miller

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. ...Continue Reading

References

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Citations

May 13, 2003·Chembiochem : a European Journal of Chemical Biology·Regula von Eggelkraut-GottankaAnnette G Beck-Sickinger
Feb 25, 2003·Protein Expression and Purification·Robert B Mackin, Meredith H Choquette

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