Comparison of enzymatic semisyntheses of peptide amides: human growth hormone releasing factor and analogs.
Bongers. J J; Offord. R E RE; Felix. A M AM; Lambros. T T; Liu. W W; Ahmad. M M; Campbell. R M RM; Heimer. E P EP
Key Findings
- A recombinant alpha‑amidating enzyme converts GRF(1‑44)‑Gly‑OH to the active GRF(1‑44)‑NH2 with near‑quantitative efficiency.
- Trypsin can amidate GRF(1‑43)‑OH (≈60% conversion) and GRF(1‑44)‑OH (≈15% conversion) in a dimethylacetamide‑leucine amide mixture.
- Direct trypsin‑catalyzed amidation of [Ala15]‑GRF(1‑29)‑OH in an ammonium acetate/ammonia buffer yields the super‑active analog at about 25% conversion.
Practical Outcomes
- For DIY peptide makers, the study suggests that using a recombinant alpha‑amidating enzyme is the most reliable way to produce fully amidated GRF. Trypsin offers a simpler, cheaper route but with modest yields, especially for the shorter 1‑29 analog, so extra purification or repeated reactions may be needed.
Summary
The paper compares different enzyme‑based ways to turn precursor forms of growth‑hormone‑releasing factor (GRF) into the active, amidated version. It shows that a recombinant enzyme can make the full‑length peptide almost completely, while trypsin can also do the job but with lower yields, especially for the shorter, super‑active 1‑29 version.
Abstract
Enzymatic semisyntheses of growth hormone releasing factor (GRF), a 44-residue peptide amide hormone, from C-terminal acid precursors, are compared. A recombinant alpha-amidating enzyme was used to convert the glycine-extended precursor, GRF(1-44)-Gly-OH, to GRF(1-44)-NH2 in an essentially quantitative fashion. Trypsin was used to convert the precursors, GRF(1-43)-OH and GRF(1-44)-OH, to GRF(1-44)-NH2 (60 and 15% conversion, respectively) in a 75% v:v N,N'-dimethylacetamide solution containing a large excess of leucine amide. Carboxypeptidase Y catalyzed transpeptidations of the precursors, GRF(1-44)-OH and [Ala44]-GRF(1-44)-OH, to GRF(1-44)-NH2 in aqueous leucine amide solutions were also attempted. The trypsin catalyzed direct amidation of [Ala15]-GRF(1-29)-OH in concentrated ammonium acetate/ammonia buffer (95% 1,4-butanediol cosolvent) to form the superactive analog, [Ala15]-GRF(1-29)-NH2 (ca. 25% conversion at equilibrium), is also described.
Study Information
pubmed
1991