Chemical Digestion of the -Asp-Cys- Sequence for Preparation of Post-translationally Modified Proteins
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Chemical Digestion of the ‑Asp‑Cys‑ Sequence for Preparation of Post‑translationally Modified Proteins Shigeru Shimamoto1 · Natsumi Mitsuoka1 · Saki Takahashi1 · Toru Kawakami2 · Yuji Hidaka1 Accepted: 30 October 2020 © Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract Numerous studies of native proteins have been reported on protein folding in this half century. Recently, post-translationally modified proteins are also focused on protein folding. However, it is still difficult to prepare such types of proteins because it requires not only the chemical but also the recombinant techniques. Native chemical ligation (NCL) is a powerful technique for producing target proteins when combined with recombinant techniques, such as expressed protein ligation (EPL). NCL basically requires an N-terminal peptide with a thioester and a C-terminal peptide which should possess a Cys residue at the N-terminus. Numerous efforts have been made to prepare N-terminal peptides carrying a thioester or a derivative thereof. However, a method for preparing C-terminal Cys-peptides with post-translational modifications has not been well developed, making it difficult to prepare such C-terminal Cys-peptides, except for chemical syntheses or enzymatic digestion. We report here on the development of a convenient technique that involves acid hydrolysis at the -Asp-Cys- sequence, to effectively obtain a C-terminal peptide fragment that can be used for any protein synthesis when combined with EPL, even under denatured conditions. Thus, this chemical digestion strategy permits the NCL strategy to be dramatically accelerated for protein syntheses in which post-translational modifications, such as glycosylation, phosphorylation, etc. are involved. In addition, this method should be useful to prepare the post-translationally modified proteins for protein folding. Keyword Native chemical ligation · Hydrolysis · Folding · Asp-Cys · Post-translational modification Abbreviations Boc tert-Butyloxycarbonyl DTT Dithiothreitol EPL Expressed protein ligation HBTU Hexafluorophosphate benzotriazole tetramethyl uranium HOBt 1-Hydroxybenzotriazole
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10930-020-09940-x) contains supplementary material, which is available to authorized users. * Shigeru Shimamoto [email protected] * Yuji Hidaka [email protected] 1
Faculty of Science and Technology, Kindai University, 3‑4‑1 Kowakae, Higashiosaka, Osaka 577‑8502, Japan
Institute for Protein Research, Osaka University, 3‑2 Yamadaoka, Suita, Osaka 565‑0871, Japan
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MALDI-TOF/MS Matrix-assisted laser desorption/ ionization time of flight mass spectrometry NCL Native chemical ligation PAGE Polyacrylamide gel electrophoresis pS Phospholyrated Ser pY Phospholyrated Tyr RNase Ribonuclease SDS Sodium dodecyl sulfate TCEP Tris(2-carboxyethyl)phosphine TFA Trifluoroacetic acid
1 Introduction More than this half century, numerous studies of native proteins have be
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