A Helping Hand to Overcome Solubility Challenges in Chemical Protein Synthesis

Abstract : Although native chemical ligation (NCL) and related chemoselective ligation approaches provide an elegant method to stitch together unprotected peptides, the handling and purification of insoluble and aggregation-prone peptides and assembly intermediates create a bottleneck to routinely preparing large proteins by completely synthetic means. In this work, we introduce a new general tool, Fmoc-Ddae-OH, N-Fmoc-1-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-3-[2-(2-aminoethoxy)ethoxy]-propan-1-ol, a heterobifunctional traceless linker for temporarily attaching highly solubilizing peptide sequences (“helping hands”) onto insoluble peptides. This tool is implemented in three simple and nearly quantitative steps: (i) on-resin incorporation of the linker at a Lys residue ε-amine, (ii) Fmoc-SPPS elongation of a desired solubilizing sequence, and (iii) in-solution removal of the solubilizing sequence using mild aqueous hydrazine to cleave the Ddae linker after NCL-based assembly. Successful introduction and removal of a Lys6 helping hand is first demonstrated in two model systems (Ebola virus C20 peptide and the 70-residue ribosomal protein L31). It is then applied to the challenging chemical synthesis of the 97-residue co-chaperonin GroES, which contains a highly insoluble C-terminal segment that is rescued by a helping hand. Importantly, the Ddae linker can be cleaved in one pot following NCL or desulfurization. The purity, structure, and chaperone activity of synthetic l-GroES were validated with respect to a recombinant control. Additionally, the helping hand enabled synthesis of d-GroES, which was inactive in a heterochiral mixture with recombinant GroEL, providing additional insight into chaperone specificity. Ultimately, this simple, robust, and easy-to-use tool is expected to be broadly applicable for the synthesis of challenging peptides and proteins.
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https://hal.archives-ouvertes.fr/hal-01407028
Contributor : Isabelle Frapart <>
Submitted on : Thursday, December 1, 2016 - 5:30:49 PM
Last modification on : Tuesday, April 2, 2019 - 5:28:03 PM

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Michael T. Jacobsen, Mark E. Petersen, Xiang Ye, Mathieu Galibert, George H. Lorimer, et al.. A Helping Hand to Overcome Solubility Challenges in Chemical Protein Synthesis. Journal of the American Chemical Society, American Chemical Society, 2016, 138 (36), pp.11775 - 11782. ⟨10.1021/jacs.6b05719⟩. ⟨hal-01407028⟩

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