Preparation of canine relaxin by Fmoc-solid phase synthesis and regioselective disulfide bond formation within the A- and B-chains

Feng Lin, Julien Tailhades, Linda J Chan, Ross Bathgate, Mohammed Akhter Hossain, John D Wade

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Background: The chemical synthesis of multi-disulfide bonded heterodimeric peptides such as insulin has long been of significant scientific and commercial interest as well as a major challenge. The development of improved protocols which includes regioselective disulfide bond formation has greatly advanced the capacity to prepare and study insulin-like peptides including canine relaxin, an important regulator of parturition and indicator of canine maternal health.

Methods: Separate, efficient solid phase synthesis of the two constituent chains (24 residue A and 35 residue B) was followed by stepwise formation of each of the three disulfide bonds, one intra within the A-chain and two interchain, by oxidation, thiolysis and iodolysis respectively.

Results: Synthetic canine relaxin having a total of 59 residues was prepared in good overall yield and shown by several criteria to be highly purified. The peptide was shown to be less potent than human relaxin (H2 relaxin) in binding to and activating the human relaxin receptor, RXFP1, in transfected cells. A circular dichrosim spectroscopic analysis showed that the canine relaxin also possessed significantly less secondary structure compared to H2 relaxin which may account for its reduced activity.

Conclusions: The synthetic protocols developed in our laboratory enabled the successful preparation of the complex, small insulinlike protein, canine relaxin. This will, in turn, allow a detailed study of both the tertiary conformation of this peptide and its role in canine reproduction.
Original languageEnglish
Article number4
Number of pages6
JournalBiochemical Compounds
Volume1
DOIs
Publication statusPublished - 2013

Keywords

  • Canine relaxin
  • H2 relaxin
  • circular dichroism spectroscopy
  • regioselective disulfide bond formation
  • RXFP1 receptor assay
  • solid phase peptide synthesis

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