Semisynthesis of human growth hormone-releasing factors by alpha-amidating enzyme catalyzed oxidation of glycine-extended precursors.

Peptide research1992PMID: 1421807

Animal StudyIn VitroSermorelin

Plain Language Summary

Scientists developed a method to produce growth hormone-releasing factor and its analogs using an alpha-amidating enzyme to convert precursor peptides into their biologically active amidated forms. The enzymatic process achieved approximately 75% yields and produced peptides with full biological potency and receptor-binding activity. This semisynthetic approach, which combines solid-phase peptide synthesis with enzymatic finishing, offers a practical route for manufacturing GHRH-based therapeutics.

Abstract

Recombinant alpha-amidating enzyme was used in the semisynthesis (1-5 mg scale) of human growth hormone-releasing factor, GRF(1-44)-NH2, by in vitro enzymatic oxidation of the glycine-extended precursor, GRF(1-44)-Gly-OH, prepared by solid-phase synthesis. The equipotent analog, GRF(1-29)-NH2, and the superactive analog, [Ala15]-GRF(1-29)-NH2, were also prepared by this route and were fully characterized. Isolated yields of about 75% were obtained, and the products each possessed full potency in an in vitro rat pituitary bioassay and full receptor-binding affinity. Methods to monitor the amidation of polypeptide substrates and analyze the final products are described, including the use of capillary zone electrophoresis. A transient alpha-hydroxyglycine intermediate, [Ala15]-GRF(1-29)-Gly(alpha-OH)-OH, was isolated and characterized. Kinetic studies with this intermediate demonstrate that the rat alpha-amidating enzyme from recombinant mouse C127 cells possesses both the monooxygenase and lyase activities needed to catalyze both steps of the amidation process.

Authors

Bongers, J; Felix, A M; Campbell, R M; Lee, Y; Merkler, D J; Heimer, E P