Semisynthesis of human growth hormone-releasing factors by alpha-amidating enzyme catalyzed oxidation of glycine-extended precursors.
Bongers. J J; Felix. A M AM; Campbell. R M RM; Lee. Y Y; Merkler. D J DJ; Heimer. E P EP
Key Findings
- Alpha‑amidating enzyme can efficiently convert a glycine‑extended GRF precursor into the active amidated peptide.
- The method produces GRF‑1‑29 and a super‑active variant with ~75% isolated yield and full biological activity in rat pituitary assays.
- A transient alpha‑hydroxyglycine intermediate was identified, confirming the enzyme performs both oxidation and lyase steps.
Practical Outcomes
- For DIY peptide makers, this study validates enzymatic amidation as a high‑yield route to active GRF‑1‑29, suggesting that using recombinant alpha‑amidating enzyme could improve purity and potency compared to chemical amidation. However, the required reagents (recombinant enzyme, solid‑phase synthesis) may be beyond typical home‑lab capabilities, so the main takeaway is that the peptide can be produced reliably if you have access to these tools.
Summary
Scientists showed a reliable way to make the growth‑hormone‑releasing peptide GRF‑1‑29 (and related versions) using an enzyme that adds an amide group. The process gives about 75% yield and the resulting peptide works just as well as the natural hormone in lab tests.
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.
Study Information
pubmed
1992