Enhancing the incorporation of lysine derivatives into proteins with methylester forms of unnatural amino acids.

We have improved the incorporation of l- and d-forms of unnatural amino acid (UAA) N-thiaprolyl-l-lysine (ThzK) into ubiquitin (UB) and green fluorescent protein (GFP) by 2-6 folds with the use of the methylester forms of the UAAs in E coli cell culture. We also improved the yields of UAA-incorporated UB and GFP with the methylester forms of N-Boc-l-Lysine (BocK) and N-propargyl-l-Lysine (PrK) by 2-5 folds compared to their free acid forms. Our work demonstrated that using methylester-capped UAAs for protein expression is a useful strategy to enhance the yields of UAA-incorporated proteins.