Generation of a bioactive neuropeptide in a cell-free system.

We have developed an in vitro assay for pre-pro-neuropeptide synthesis and processing. Mouse proopiomelanocortin (POMC) cDNA was cloned into a vector containing T7 promoter. In vitro transcription and translation were carried out to produce the proopiomelanocortin peptide. The pro-peptide was processed by incubating with cell extract of mouse and rat pituitary cell lines. The activity of processed mature peptide was tested in a cell line expressing human melanocortin 4 receptor (MC4R). Using this approach, we produced biologically active alpha-melanocyte-stimulating hormone (alpha-MSH). Furthermore, we developed a universal functional assay for G-protein-coupled receptors (GPCRs) using a reporter gene assay. More than 20 different GPCRs were examined using this functional assay. Our results demonstrated that the activity of all GPCRs could be measured by the functional assay. It should be possible to identify novel bioactive peptides for orphan GPCRs by the combination of in vitro processing and GPCR functional assays.