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 proteolyti c processing of prohormones and proproteins into active peptide hormon es and neurotransmitters. However, the lack of prohormone proteins has been a limiting factor in elucidating proteolytic mechanisms for conv ersion of prohormones into active peptides. Therefore, in this study, cloned cDNAs encoding the prohormones proenkephalin (PE), pro-neuropep tide Y (pro-NPY), proopiomelanocortin (POMC), and beta-protachykinin ( beta-PT) were utilized to express recombinant prohormones in Escherich ia coil. High-level expression of milligrams of prohormones was achiev ed with the pET3c expression vector utilizing the T7 promoter for prod uction of PE, pro-NPY, and POMC, as demonstrated by SDS-PAGE gel elect rophoresis, Western blots, and S-35-methionine labeling. In addition, beta-PT was expressed at high levels as fusion proteins with the malto se-binding protein and glutathione S-transferase by the pMAL-c and pGE X-2T expression vectors, respectively. Relative rates of processing by the established processing proteases ''prohormone thiol protease'' (P TP), 70-kDa aspartyl protease, and PC1/3 and PC2 (PC, prohormone conve rtase) were examined with purified PE, pro-NPY, and POMC. Distinct pre ferences of processing enzymes for different prohormones was demonstra ted. PTP prefered PE and pro-NPY substrates, whereas little processing of POMC was detected. In contrast, the 70-kDa aspartyl protease cleav ed 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 re combinant prohormones for in vitro processing studies. (C) 1997 Academ ic Press.