The substrate profile of peptiligase, a stable enzyme designed for peptide ligation in aqueous environments, was mapped using six different peptide libraries. The most discriminating substrate binding pocket proved to be the first nucleophile binding subsite (S1), which is crucial for the peptide ligation yield. Two important amino acids shaping the S1 pocket are M213 and L208. A site-saturation library of the M213 position yielded two variants with a significantly broadened substrate profile, i.e., M213G and M213P. Next, examination of two libraries with M213G+L208X and M213P+L208X (with X being any proteinogenic amino acid) resulted in a toolbox of enzymes which can accommodate any proteinogenic amino acid in the S1 pocket, except proline. The applicability of a particular enzyme variant in chemoenzymatic peptide synthesis was demonstrated by coupling at the gram scale of two peptide segments to yield exenatide, a 39-mer therapeutic peptide used in the treatment of diabetes type II. The overall yield of 43% is at least 2-fold higher than yields reported for conventional syntheses of exenatide by full solid-phase peptide synthesis; large-scale production costs are expected to be significantly reduced if the enzymatic coupling process is employed to manufacture this peptide.
- chemoenzymatic peptide synthesis
- genetic engineering
- peptide ligase
- segment condensation