ABSTRACT
The use of light to control protein function is a critical tool in chemical biology. Here we describe the addition of a photocaged histidine to the genetic code. This unnatural amino acid becomes histidine upon exposure to light and allows for the optical control of enzymes that utilize active-site histidine residues. We demonstrate light-induced activation of a blue fluorescent protein and a chloramphenicol transferase. Further, we genetically encoded photocaged histidine in mammalian cells. We then used this approach in live cells for optical control of firefly luciferase and, Renilla luciferase. This tool should have utility in manipulating and controlling a wide range of biological processes.
Subject(s)
Amino Acids , Histidine , Animals , Histidine/genetics , Amino Acids/chemistry , Proteins/metabolism , Luciferases, Renilla/genetics , Genetic Code , Mammals/genetics , Mammals/metabolismABSTRACT
We have improved the incorporation of l- and d-forms of unnatural amino acid (UAA) Nε-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 Nε-Boc-l-Lysine (BocK) and Nε-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.