Absolute Quantitation of Non-Standard Amino Acids in Proteins Guiding the Evolution of Orthogonal Translation Systems

摘要

Programmed insertion of non-standard amino acids (NSAAs) into proteins provides an elegant strategy for encoding post-translational modifications or amino acids with diverse bio-orthogonal chemistries into proteins. This technology relies on an orthogonal pair of an aminoacyl-tRNA synthetases (aaRS) and a tRNA designed to recognize a specific codon such as UAG. By encoding one or multiple UAG codons, site-specific incorporation of the NSAA at the designated site can be accomplished. We have engineered release factor 1 deficient, genomically recoded E. coli bacteria that provide a dedicated UAG codon for NSSA incorporation1. This strain has increased protein yields for the incorporation of single NSAAs into proteins but multi-site incorporation of a NSAA into a protein remains difficult. The low activity of aaRS used for NSAA incorporation has been identified as a potential source of the problem. We were wondering whether we could improve multi-site NSAA incorporation by engineering aaRS variants with improved performance. Here we describe a synthetic biology approach for the evolution of several novel aaRS variants for insertion of pAcF and perform in-depth analysis of the produced protein by label free proteomics and MRM MS. Using quantitative mass spectrometry we demonstrate that the newly evolved aaRS variants can insert up to 30 instances of the NSAA pAcF into GFP.