A Leucyl-tRNA Synthetase Without the Editing Domain and its Directed Evolution for Unnatural Protein Engineering

Abstract

This project aims to develop a functional leucine-tRNA synthetase (LeuRS) without the editing domain and perform its directed evolution to incorporate various unnatural amino acids with desirable properties in Escherichia coli. This method will be useful to generate proteins with new or enhanced properties and will also serve as a powerful tool to study structure and function of proteins. We generated billions of different variants of the Methanobacterium thermoautotrophium LeuRS (MLRS) whose editing domain was replaced by peptide linkers containing six randomized amino acid residues. A genetic selection was subsequently performed to obtain a functional LeuRS without the editing domain (MLRS DCP1). The best variant was used in the subsequent directed evolution experiment. From the structure-based sequence alignment with a bacterial LeuRS, the leucine binding site of the MLRS was identified. The five amino acids found in the leucine binding pocket were randomized to generate billions of different variants of MLRS DCP1. In order to select MLRS DCP1 variants specific for unnatural amino acids, a dual genetic screen will be performed. In the positive selection, the bacteria cells containing MLRS DCP1 capable of attaching any natural or unnatural amino acid onto the tRNA can survive in the presence of chloramphenicol. In negative selection in the absence of the unnatural amino acid, the cells containing the MLRS DCP1 variants that add any natural amino acids to the tRNA die in the presence of 5-Fluorouracil. The cells harboring the MLRS DCP1 variants that attach only unnatural amino acids onto the tRNA can survive on 5-FU. These successful variants will be tested for the effective incorporation of the unnatural amino acid with our model protein called the Z-domain and for specific intended applications with various proteins.