Enabling chemical protein (semi)synthesis via reducible solubilizing tags (RSTs)

Abstract

Chemical synthesis of proteins with poor solubility presents a challenging task. The existing solubilizing tag strategies are not suitable for the expressed protein segment. To address this issue, we report herein that solubilizing tags could be introduced at the side chain of the peptide and C-terminal peptide salicylaldehyde esters via a disulfide linker. Such reducible solubilizing tags (RSTs) are compatible with peptide salicylaldehyde ester-mediated Ser/Thr ligation and Cys/Pen ligation for purifying and ligating peptides with poor solubility. This strategy features operational simplicity and readily accessible materials. Both the protein 2B4 cytoplasmic tail and FCER1G protein have been successfully synthesized via this strategy. Of particular note, the RST strategy could be used for solubilizing the expressed protein segment for protein semi-synthesis of the HMGB1 protein.

Fig. 1. General strategy of reducible solubilizing tags (RSTs).

Fig. 2. Chemical ligation of model peptides with different solubilizing tags attached at internal Cys via the RST strategy. (a) General route of chemical ligation between the model peptide salicylaldehyde ester with RSTs at internal Cys and model N-terminus peptide. (b) UPLC trace following the CPL between the model peptide salicylaldehyde ester without solubilizing tags and with different RSTs at internal Cys and model N-terminus peptide. N,S-benzylidene acetals 3a–e are diastereomeric mixtures. (c) Scope of RSTs at internal Cys incorporated with CPL.

Fig. 3. Second generation of RSTs. (a) General synthesis route of the peptide salicylaldehyde ester with reducible solubilizing tags at salicylaldehyde. (b) General route of chemical ligation between the model peptide salicylaldehyde ester with RSTs via the salicylaldehyde ester and model N-terminus peptide. (c) UPLC trace following CPL between the model peptide salicylaldehyde ester with different RSTs via the salicylaldehyde ester and model N-terminus peptide. (d) Scope of RSTs via salicylaldehyde ester incorporated with CPL. N,S-Benzylidene acetals 3g–f are diastereomeric mixtures.

Fig. 4. Synthesis of the protein 2B4 cytoplasmic tail. (a) Sequence of the protein 2B4 cytoplasmic tail. (b) Synthesis route of the protein 2B4 cytoplasmic tail. (c) UPLC trace following the 1^st STL between peptide 6 and 7; peak (A): hydrolysis of 7. (d) UPLC trace following peptide 8 acidolysis and the Thz opening step. (e) UPLC trace following the 2^nd CPL and acidolysis step between peptide 9 and 10; peak (B): the thioacetal product of 10 and EDT. (f) UPLC trace following the 3^rd STL, acidolysis and desulfurization step between peptide 11 and 12, and purified 13; peak (C): hydrolysis of 12. (g) ESI-MS of purified 13.

Fig. 5. Synthesis of membrane protein FCER1G. (a) Sequence of membrane protein FCER1G. (b) Synthesis route of membrane protein FCER1G. (c) UPLC trace following CPL, acidolysis and the reduction step between peptide 14 and 15; peak (A): dimer of 14; peak (B): hydrolysis of 15; peak (C): hydrolysis of 15 without the K₁₀ tag. 16a is a diastereomeric mixture. (d) ESI-MS image of purified 16.

Fig. 6. Synthesis of protein HMGB1. (a) Sequence of protein HMGB1. (b) Synthesis route of protein HMGB1. (c) UPLC trace of purified 18 and purified 19. (d) UPLC trace following STL, acidolysis and the reduction step between 19 and 20; peak (A): hydrolysis of 20 without the biotin tag; peak (B): 20 without the biotin tag. (e) Deconvolution of ESI-MS of purified 21. (f) SDS-Page analysis of 18, 19 and 21; the gel was visualized by Coomassie blue staining. Non-reductive: the loading buffer contains non-reductive additives. Reductive: the loading buffer contains 100 mM DTT.