Production and biological activity of hybrid growth hormone-releasing hormone propeptides

Growth hormone-releasing hormone (GHRH), a hypothalamic hormone that stimulates the synthesis and release of growth hormone (GH) from anterior pituitary cells, has been previously produced by synthetic peptide chemistry and recombinant DNA procedures. GHRH is capable of stimulating growth as well as eliciting other anabolic effects on animals and thus may have potential applications in agriculture and human medicine. However, economical production of GHRH by recombinant DNA process has been difficult since GHRH is degraded rapidly by endogenous E. coli proteases. We report here an efficient process to produce hybrid GHRH analogs of higher molecular weight. These hybrid GHRH propeptides (proGHRH) are comprised of an analog of GHRH (44 aa) and the human GHRH carboxy-terminal peptide (33 aa). In E. coli K-12 RV308, the expression levels of the proGHRH analogs were estimated to be 10% of the total cellular protein. An in vitro assay to measure the release of rat growth hormone by GHRH analogs using crude E. coli lysates was also developed. This assay showed that the proGHRH analogs produced in E. coli efficiently stimulated GH release from rat anterior pituitary cells. One proGHRH analog, [alao]-proGHRH, was purified ans shown to efficiently elevate plasma GH levels in wether lambs. Our data indicate that the hybrid proGHRH peptides, unlike other hormone propeptides such as proinsulin, are remarkably bioactive.