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.
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Abbreviations
- Boc:
-
tert-Butyloxycarbonyl
- DTT:
-
Dithiothreitol
- EPL:
-
Expressed protein ligation
- HBTU:
-
Hexafluorophosphate benzotriazole tetramethyl uranium
- HOBt:
-
1-Hydroxybenzotriazole
- 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
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Acknowledgements
We wish to thank Prof. Hironobu Hojo, Osaka University for peptide synthesis. This work was partially supported by JSPS KAKENHI Grant number 16K01925 and Grants-in-Aid for Young Scientists (B) Grant number 16K18868.
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NM, ST, and TK performed the experiments with assistance from SS and YH; SS analyzed the data and made suggestions; YH and SS supervised the project. All of the authors participated in the writing of the manuscript.
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Shimamoto, S., Mitsuoka, N., Takahashi, S. et al. Chemical Digestion of the -Asp-Cys- Sequence for Preparation of Post-translationally Modified Proteins. Protein J 39, 711–716 (2020). https://doi.org/10.1007/s10930-020-09940-x
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DOI: https://doi.org/10.1007/s10930-020-09940-x