An Enzyme‐Cleavable Solubilizing‐Tag Facilitates the Chemical Synthesis of Mirror‐Image Proteins

Abstract

Mirror‐image proteins (D‐proteins) are useful in biomedical research for purposes such as mirror‐image screening for D‐peptide drug discovery, but the chemical synthesis of many D‐proteins is often low yielding due to the poor solubility or aggregation of their constituent peptide segments. Here, we report a Lys‐C protease cleavable solubilizing‐tag and its use to synthesize difficult‐to‐obtain D‐proteins. Our tag is easily installed onto multiple amino acids such as D‐Lys, D‐Ser, D‐Thr and/or the N‐terminal D‐amino acid of hydrophobic peptides, is impervious to various reaction conditions, such as peptide synthesis, ligation, desulfurization, and transition metal‐mediated deprotection, and yet can be completely removed by Lys‐C protease under denaturing conditions to give the desired D‐protein. The efficacy and practicality of the new method were exemplified in the synthesis of two challenging D‐proteins: D‐enantiomers of PD‐1 IgV domain and SARS‐CoV‐2 envelope protein, in high yield. This work demonstrates that the enzymatic cleavage of solubilizing tags under denaturing conditions is feasible, thus paving the way for the production of more challenging D‐proteins.

Chemical Synthesis of Human Proteoforms and Application in Biomedicine

“…The methods of temporary structural support to promote protein ligation and folding, along with other tools such as isoacyl dipeptides − and removable solubilizing tags, − have expanded the capabilities of chemical protein synthesis, providing effective approaches for synthesizing increasingly challenging and larger protein targets. These advancements make it more effective to synthesize midsized human proteoforms with lengths of 300–500 amino acids, including those with poor water solubility (e.g., membrane proteins) …”