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Butelase-mediated cyclization and ligation of peptides and proteins

Nature Protocols volume 11, pages 1977–1988 (2016)Cite this article

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Abstract

Enzymes that catalyze efficient macrocyclization or site-specific ligation of peptides and proteins can enable tools for drug design and protein engineering. Here we describe a protocol to use butelase 1, a recently discovered peptide ligase, for high-efficiency cyclization and ligation of peptides and proteins ranging in size from 10 to >200 residues. Butelase 1 is the fastest known ligase and is found in pods of the common medicinal plant Clitoria ternatea (also known as butterfly pea). It has a very simple C-terminal-specific recognition motif that requires Asn/Asp (Asx) at the P1 position and a dipeptide His–Val at the P1′ and P2′ positions. Substrates for butelase-mediated ligation can be prepared by standard Fmoc (9-fluorenylmethyloxycarbonyl) chemistry or recombinant expression with the minimal addition of this tripeptide Asn–His–Val motif at the C terminus. Butelase 1 achieves cyclizations that are 20,000 times faster than those of sortase A, a commonly used enzyme for backbone cyclization. Unlike sortase A, butelase is traceless, and it can be used for the total synthesis of naturally occurring peptides and proteins. Furthermore, butelase 1 is also useful for intermolecular ligations and synthesis of peptide or protein thioesters, which are versatile activated intermediates necessary for and compatible with many chemical ligation methods. The protocol describes steps for isolation and purification of butelase 1 from plant extract using a four-step chromatography procedure, which takes ∼3 d. We then describe steps for intramolecular cyclization, intermolecular ligation and butelase-mediated synthesis of protein thioesters. Butelase reactions are generally completed within minutes and often achieve excellent yields.

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Figure 1: Schematic concepts and proposed applications of butelase 1 (center) in protein engineering and biological research.
Figure 2: Schematic presentation of butelase-mediated ligation.
Figure 3: Protein thioester preparation using butelase-mediated ligation.
Figure 4: Butelase-mediated cyclization of human hormonal peptides.
Figure 5: An example of butelase-mediated cyclization of GFP.
Figure 6: Butelase-mediated intermolecular ligation of KALVINHV and GIGGIR.
Figure 7: An example of butelase-mediated thioesterification of ubiquitin.

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Acknowledgements

This work was supported by NTU iFood research grant M4081467.080 and Singapore National Research Foundation grant NRFCRP8-2011-05 to J.P.T.

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Authors and Affiliations

  1. School of Biological Sciences, Nanyang Technological University, Singapore

    Giang K T Nguyen, Yibo Qiu, Yuan Cao, Xinya Hemu, Chuan-Fa Liu & James P Tam

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  1. Giang K T Nguyen
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  2. Yibo Qiu
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  3. Yuan Cao
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Contributions

G.K.T.N. drafted the manuscript. Y.Q. and X.H. developed the protocol for intermolecular ligation. Y.C. and C.-F.L. developed the protocol for butelase-mediated protein thioester synthesis. J.P.T. conceived the idea, supervised the project and revised the manuscript.

Corresponding author

Correspondence to James P Tam.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Chromatogram of the C. ternatea extract by preparative anion exchange.

Fractions containing ligase activity are indicated by the double arrow. The right axis indicate the percentage of the elution buffer. The cyan line indicates the gradient used for HPLC separation.

Supplementary Figure 2 A typical MS-guided screening of ligase-containing fractions.

The linear precursor of SFTI has a m/z value of 1766.9 which is converted to 1512.7 in the presence of butelase 1.

Supplementary Figure 3 Chromatogram of the C. ternatea extract by size exclusion chromatography.

Fractions containing ligase activity are indicated by the double arrow.

Supplementary Figure 4 Chromatogram of butelase 1 separation by analytical anion exchange.

Fractions containing ligase activity are indicated by the double arrow. The cyan line indicates the gradient used for HPLC separation.

Supplementary Figure 5 SDS-PAGE analysis of purified butelase 1.

The gel was stained with Coomassie blue. Purified butelase 1 should migrate as a single band with a molecular weight of about 37 kDa.

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Supplementary Figures 1–5 (PDF 673 kb)

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Nguyen, G., Qiu, Y., Cao, Y. et al. Butelase-mediated cyclization and ligation of peptides and proteins. Nat Protoc 11, 1977–1988 (2016). https://doi.org/10.1038/nprot.2016.118

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