A cysteine-specific solubilizing tag strategy enables efficient chemical protein synthesis of difficult targets

Abstract

We developed a new cysteine-specific solubilizing tag strategy via a cysteine-conjugated succinimide. This solubilizing tag remains stable under common native chemical ligation conditions and can be efficiently removed with palladium-based catalysts. Utilizing this approach, we synthesized two proteins containing notably difficult peptide segments: interleukin-2 (IL-2) and insulin. This IL-2 chemical synthesis represents the simplest and most efficient approach to date, which is enabled by the cysteine-specific solubilizing tag to synthesize and ligate long peptide segments. Additionally, we synthesized a T8P insulin variant, previously identified in an infant with neonatal diabetes. We show that T8P insulin exhibits reduced bioactivity (a 30-fold decrease compared to standard insulin), potentially contributing to the onset of diabetes in these patients. In summary, our work provides an efficient tool to synthesize challenging proteins and opens new avenues for exploring research directions in understanding their biological functions.

Fig. 1. Illustration of different solubilizing tag strategies. (A) Temporary solubilization tag after NCL. (B) Temporary solubilization tag before ligation. (C) Structure and properties of the maleimide-based zwitterionic solubilizing tag. (D) Conjugation and removal mechanism of the tag.

Fig. 2. Cysteine-conjugated Su-(EK)_n solubilizing tag strategy applied to model peptides with poor solubility. (A) Solubilization test of the hydrophobic IL-2 C-terminal peptide (1) and conditions screening for solubilizing tag removal. (B) Solubilization test of the hydrophobic mini-insulin A chain (3) and conditions screening for solubilizing tag removal.

Fig. 3. Structure and synthesis of IL-2. (A) Sequence of IL-2. (B) Synthetic route of IL-2. (C) Solubility of peptide 8 in various solvents. (D) HPLC traces of 6-MPAA and 7 during first NCL reaction (* = hydrolyzed and cyclized 6-MPAA). (E) HPLC traces of 9 and 10 during second NCL reaction (** = hydrolyzed and cyclized 9). (F) HPLC traces for palladium-catalyzed removal of the solubilizing tags (the double peaks of 2, 10 and 11 on the HPLC are due to chiral isomers). (G) Enhancing solubility of challenging peptide 10 with Mal-(EK)₅versus Mal-(K)₁₀ tags in neutral ligation buffer. (H) Solubility of peptides 8, 9a (Seg 1-2-Su-K₆), 9b (Seg 1-2-Su-K₁₀), and 9 in ligation buffer and HPLC buffer.

Fig. 4. Characterization of synthetic IL-2. (A) Mass spectra traces of linear IL-2 (12) and folded IL-2. (B) HPLC trace of linear IL-2 (12) folding to folded IL-2. (C) Dose–response of HEK-Blue™ IL-2 cells to recombinant IL-2 (rIL-2) and synthetic IL-2 (sIL-2).

Fig. 5. Structure and synthesis of T8P insulin. (A) Sequence and crystal structure of human insulin (PDB code 5CNY) and T8P insulin, with point mutation indicated in red. (B) Synthetic route of T8P insulin. (C) Physical states and HPLC traces of 13 before and after reaction with Mal-(EK)₃, yielding 14. (D) Mass spectrum of T8P insulin.

Fig. 6. Comparative evaluation of activity and structural differences between T8P insulin and human insulin. (A) Insulin signaling activation (AKT phosphorylation) assay of T8P insulin and human insulin. (B) CD spectra of T8P insulin and human insulin. (C) Overlap of T8P insulin (predicted by AlphaFold) and human insulin structures. (D and E) Structures of human insulin and T8P insulin, along with their A chain N-terminus conformation. The yellow dashed lines represent hydrogen bonds, and the pink dashed lines indicate the corresponding atomic distances.