Site-specific protein modification by genetic encoded disulfide compatible thiols

Cysteine chemistry provides a low cost and convenient way for site-specific protein modification. However, recombinant expression of disulfide bonding containing protein with unpaired cysteine is technically challenging and the resulting protein often suffers from significantly reduced yield and activity. Here we used genetic code expansion technique to introduce a surface exposed self-paired di-thiol functional group into proteins, which can be selectively reduced to afford active thiols. Two compounds containing self-paired disulfides were synthesized, and their genetic incorporations were validated using green fluorescent proteins (GFP). The compatibility of these self-paired di-thiols with natural disulfide bond was demonstrated using antibody fragment to afford site-specifically labeled antibody. This work provides another valuable building block into the chemical tool-box for site-specific labeling of proteins containing internal disulfides.