Testing Of The Mutual Orthogonality Of The Chimeric System And Its Application

Post-translational modification(PTM)of proteins plays a vital role in the regulation of protein functions and cellular biological process.Here,a variety of methods were set up and applied to explore two chemical biology toolkits,genetic code expanding(GCE)and Lb^pro*,for studying PTM.GCE employs orthogonal aminoacyl-tRNA synthetase/tRNA pairs,which recognizes the site-specific codon and inserts unnatural amino acids(UAA)with versatile properties on the target proteins.Therefore,GCE is a promising approach in the current research for PTM.However,the lack of orthogonal translation systems limits the application in biological research.Here,chimeric phenylalanine translation system,paired with Ma.system or Mj.system,was constructed and applied to introduce UAA into green fluorescent protein(GFP).Correct insertion of respective UAA verified via fluorescence screening,western-blotting and LC/MS,proves the mutual orthogonality between the chimeric phenylalanine and Ma.system or Mj.system.Hence,the scope of orthogonal translation systems available is expanded,and the application of the chimeric system in the research of protein post-translational modification is broadened.Lb^pro*is a protease encoded by the foot-and-mouth disease virus,containing L102W mutation.Lb^pro*can convert the ubiquitylome into glycine-glycine dipeptide modified proteome.With the combination of LC/MS,this toolkit is capable to quantify the different branched ubiquitin in the polyubiquitin chain,further mapping the profile of the structure of the polyubiquitin chain.However,due to its low recognition specificity and hydrolytic activity of ubiquitin chain,Lb^pro*is limited for wide application of ubiquitin research.Here,protein rational design and directed evolution were used for Lb^pro*engineering.First,a high-throughput screening system was constructed based on the fluorescence resonance energy transfer(FRET)principle.After analysis of the interface between Lb^pro*and ubiquitin,L143 and L178 were chosen for saturation mutagenesis library construct and subjected to a high-throughput screen.Then,through docking and redesign of the Rosetta algorithm,site-directed mutation of Lb^pro*was performed.Site 106 and 131 were confirmed to improve the hydrolytic activity.Finally,7 analogues of tryptophan were introduced at the L102 position through the chimeric system to explore the mechanism of site L102.