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Protein chemical synthesis by α-ketoacid–hydroxylamine ligation


Total chemical synthesis of proteins allows researchers to custom design proteins without the complex molecular biology that is required to insert non-natural amino acids or the biocontamination that arises from methods relying on overexpression in cells. We describe a detailed procedure for the chemical synthesis of proteins with the α-ketoacid–hydroxylamine (KAHA ligation), using (S)-5-oxaproline (Opr) as a key building block. This protocol comprises two main parts: (i) the synthesis of peptide fragments by standard fluorenylmethoxycarbonyl (Fmoc) chemistry and (ii) the KAHA ligation between fragments containing Opr and a C-terminal peptide α-ketoacid. This procedure provides an alternative to native chemical ligation (NCL) that could be valuable for the synthesis of proteins, particularly targets that do not contain cysteine residues. The ligation conditions—acidic DMSO/H2O or N-methyl-2-pyrrolidinone (NMP)/H2O—are ideally suited for solubilizing peptide segments, including many hydrophobic examples. The utility and efficiency of the protocol is demonstrated by the total chemical synthesis of the mature betatrophin (also called ANGPTL8), a 177-residue protein that contains no cysteine residues. With this protocol, the total synthesis of the betatrophin protein has been achieved in around 35 working days on a multimilligram scale.

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Figure 1: General description of the KAHA ligation.
Figure 2: Synthesis of betatrophin protein by sequential KAHA ligations.
Figure 3: Characterization of betatrophin 9.
Figure 4: RP–HPLC monitoring of the KAHA ligation–oxidation in one pot of H2N-F1(22–59)-Leu-KA 1 with Opr-F2(62–95)-Leu-SY 2.
Figure 5: RP–HPLC monitoring of the KAHA ligation–Fmoc deprotection and O–N shift in one pot of FmocOpr-F4(135–165)-Leu-KA 3 with Opr-F5(168–198)-COOH 4.


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This work was supported by the ETH Zürich and the Swiss National Science Foundation (200020_150073). We thank the LOC MS Service for analyses and T. Hayashi for his help with folding and CD spectrum measurement.

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Authors and Affiliations



T.J.H. and C.E.M. performed the experiments, compound characterization and data analysis. All authors contributed to experimental design, discussions and writing of the manuscript.

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Correspondence to Jeffrey W Bode.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 SDS-PAGE and CD spectra of purified betatrophin 9

(a) SDS-PAGE/Coomassie staining analysis of purified synthetic betatrophin 9. (b) circular dichroism spectra of refolded synthetic betatrophin 9. ~20μM of betatrophin 9 in PBS buffer pH 7.2 was analyzed in a 1 mm quartz cell.

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Supplementary Figure 1, Supplementary Data 1–9 (PDF 2286 kb)

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Harmand, T., Murar, C. & Bode, J. Protein chemical synthesis by α-ketoacid–hydroxylamine ligation. Nat Protoc 11, 1130–1147 (2016).

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