Omniligase-1 for Efficient Peptide Ligation

A modified version of subtilisin from B. subtilis, Omniligase-1 (developed by EnzyPep B.V.) has been selected to ligate side-chain unprotected peptide segments. Unlike classical Native Chemical Ligation, the approach using Omniligase is not limited to fragments bearing an N-terminal cysteine.

Omniligase-1 catalyzes the reaction between an ester fragment (acyl donor) and an amine (acyl acceptor, nucleophile) fragment. The enzyme can be used to either cyclize a single peptide ester fragment, or to ligate two fragments. In the latter case, the N-terminus of the peptide ester fragment has to be protected in order to avoid cyclization. Ligation proceeds under mild aqueous conditions without racemization, with ligation yields as high as 98.9%.

Broad Substrate Specificity

Due to its broad substrate specificity, Omniligase-1 recognizes many different kinds of acyl donors. Alkyl-, aryl- and thioesters can all be used as peptide ester fragment. However, we recommend carboxyamidomethyl (Cam) esters, or substituted derivatives thereof (e.g. –Cam-Leu-OH or –Cam-Leu-NH2). Peptide Cam-esters (and derivatives) can be prepared by standard solid-phase techniques. If necessary, the polarity and hence solubility of the peptide ester fragment can be tuned by changing the type of C-terminal ester.

For ligation of two fragments, the peptide ester fragment must bear an N-terminal protecting group to avoid unwanted cyclization. For this purpose, we recommend the phenylacetyl group, which can be removed under mild conditions using commercially available acylases (e.g. immobilized PenG amidase from Iris Biotech: EZ60030, EZ60040).

The amine (acyl acceptor, nucleophile) fragment requires no further modifications and can be used completely unprotected.

Choosing your ligation site

Omniligase-1 has six substrate recognition subsites: four at the acyl donor binding site (S1-S4), and two at the amine site (S1' and S2'). The substrate scope of omniligase-1, which has been completely determined, is very broad.

With the exception of proline in the S1, S2, S1’ and S2’sites, the enzyme tolerates every amino acid in all of the substrate binding sites. The ligation yield largely depends on the nature of the amino acids in the S1’ and S2’ pockets. Asp and Glu should be avoided at the S2’ position, because ligation will result in lower yield. In summary there are many options of amino acid fragment combinations which enable ligation yields of over 70%.

Notice:

OMNI Kit components may be used for research purposes only. They may not be used for any other purpose, including, but not limited to, use in drugs, in vitro diagnostics purposes, therapeutics or in humans. EnzyPep products may not be transferred to third parties, sold, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without written approval of EnzyPep BV.

References

• Peptiligase, an Enzyme for Efficient Chemoenzymatic Peptide Synthesis and Cyclization in Water; A. Toplak, T. Nuijens, P. J. L. M. Quaedflieg, B. Wu and D. B. Janssen; Advanced Synthesis & Catalysis 2016; 358: 2140-2147. doi:10.1002/adsc.201600017
• Improving the carboxyamidomethyl ester for subtilisin A-catalysed peptide synthesis; R. J. A. C. de Beer, T. Nuijens, L. Wiermans, P. J. L. M. Quaedflieg and F. P. J. T. Rutjes; Organic & Biomolecular Chemistry 2012; 10: 6767-6775. doi:10.1039/c2ob25662b
• Synthesis of proteins by subtiligase; A. C. Braisted, J. K. Judice and J. A. Wells; Methods in enzymology 1997; 289: 298-313.
• A designed peptide ligase for total synthesis of ribonuclease A with unnatural catalytic residues; D. Jackson, J. Burnier, C. Quan, M. Stanley, J. Tom and J. Wells; Science 1994; 266: 243-247. doi:10.1126/science.7939659