Aminoacyl-tRNA synthetases:investigations of tRNA specificity for application in ProxiMAX/synthetic biology

Abstract

ProxiMAX randomisation is a nondegenerate saturation mutagenesis technology that offers control over identity, location and relative ratio of amino acids within a protein library. It employs a maximum of 20 codons in saturated positions to encode 20 amino acids. This eliminates bias, degeneracy and provides maximal diversity. Since a maximum of 20 codons are used, many codons remain available to encode unnatural amino acids (UAAs). Incorporation of UAAs is essential tool in protein engineering to expand protein repertoires. The aim of this project is to establish fundamentals for the development of an in vitro transcription/translation system that will ultimately be coupled with ProxiMAX randomisation to produce synthetic gene libraries simultaneously encoding multiple UAAs.The focus of the current project was E. coli Alanyl-tRNA synthetase (AlaRS), an enzyme that aminoacylates tRNAAla with L-Alanine. The aim was to engineer the active site of the synthetase to incorporate UAAs and to develop an assay by which the resulting AlaRS protein library might be screened. ProxiMAX randomisation was employed to produce two variant libraries of AlaRS, each had removed native amino acid editing function. Six positions from amino acid binding pocket were selected for randomisation to encode 18 natural amino acids. Each resulting library encoded >107 novel variant proteins.The initial development of screening assay for synthetase libraries involved using eGFP with stop codon as a reporter gene to assess aminoacylation of tRNA suppressor by AlaRS enzyme. The assay was designed in S. cerevisiae to prevent exogenous E. coli AlaRS and tRNA from working with endogenous system. The controls designed for the assay revealed that yeast read through the stop codon in eGFP and therefore, aminoacylation of tRNA suppressor by AlaRS proved to be difficult to assess. Alternative stratagems for future screens are considered in light of the results contained within the current study.