Applications of genetic code expansion technology in eukaryotes

Unnatural amino acids(UAAs)have gained significant attention in protein engineering and drug development owing to their ability to introduce new chemical functionalities to proteins.In eukaryotes,genetic code expansion(GCE)enables the incorporation of UAAs and facilitates posttranscriptional modification(PTM),which is not feasible in prokaryotic systems.GCE is also a powerful tool for cell or animal imaging,the monitoring of protein interactions in target cells,drug development,and switch regulation.Therefore,there is keen interest in utilizing GCE in eukaryotic systems.This review provides an overview of the application of GCE in eukaryotic systems and discusses current challenges that need to be addressed.

Therapeutic applications of genetic code expansion

In nature,a limited,conservative set of amino acids are utilized to synthesize proteins.Genetic code expansion technique reassigns codons and incorporates noncanonical amino acids(ncAAs)through orthogonal aminoacyltRNA synthetase(aaRS)/tRNA pairs.The past decade has witnessed the rapid growth in diversity and scope for therapeutic applications of this technology.Here,we provided an update on the recent progress using genetic code expansion in the following areas:antibody-drug conjugates(ADCs),bispecific antibodies(BsAb),immunotherapies,long-lasting protein therapeutics,biosynthesized peptides,engineered viruses and cells,as well as other therapeutic related applications,where the technique was used to elucidate the mechanisms of biotherapeutics and drug targets.

Light-controlled phosphorylation in the TrkA-Y785 site by photosensitive UAAs activates the MAPK/ERK signaling pathway

Background:This paper aims to establish a light-controlled phosphorylation detection method at the Y785 site of tropomyosin receptor kinase A(TrkA)receptor in mammalian cells by using genetic code expansion technology and detecting the effects of optical activation of this site on the downstream MAPK/ERK pathway.The study is based on the current situation that the regulatory mechanism of TrkA phosphorylation has not been fully elucidated.Methods:Two photosensitive unnatural amino acids,p-azido-L-phenylalanine(AzF)and photo-caged tyrosine(ONB)were introduced into the TrkA-Y785 site by genetic code expansion technology and site-directed mutagenesis.Western blotting and laser confocal imaging were conducted to analyze the effects of this site on activating the MAPK/ERK pathway and nerve cell differentiation before and after photostimulation.Results:Our results supplemented the light-controlled results of the TrkA-Y785 site based on our previous research and verified that Y785 also makes important contributions in regulating the MAPK/ERK pathway.Conclusion:This study demonstrated the significant contributions of the TrkAY785 site in regulating the ERK pathway by precisely controlling the phosphorylation state of a single tyrosine site.

Site-specific protein modification by genetic encoded disulfide compatible thiols

Cysteine chemistry provides a low cost and convenient way for site-specific protein modification.However,recombinant expression of disulfide bonding containing protein with unpaired cysteine is technically challenging and the resulting protein often suffers from significantly reduced yield and activity.Here we used genetic code expansion technique to introduce a surface exposed self-paired dithiol functional group into proteins,which can be selectively reduced to afford active thiols.Two compounds containing self-paired disulfides were synthesized,and their genetic incorporations were validated using green fluorescent proteins(GFP).The compatibility of these self-paired di-thiols with natural disulfide bond was demonstrated using antibody fragment to afford site-specifically labeled antibody.This work provides another valuable building block into the chemical tool-box for site-specific labeling of proteins containing internal disulfides.

Genetic Encoding of a Photocaged Glutamate for Optical Control of Protein Functions

Genetic encoding of photocaged noncanonical amino acids provides a powerful tool to study protein functions through optical control but is not yet available for acidic amino acids.Herein,we report the first site-specific genetic encoding of a photocaged glutamate,4-methoxy-7-nitroindolinyl caged glutamate(MNI-Glu),into recombinant proteins via an expanded genetic code through evolved EcLeuRS/tRNA pair.Using two enzymes as examples,we demonstrate that substituting the conserved-active-site glutamate of a secreted alkaline phosphatase and a protease HRV3C to MNI-Glu allows photoregulatory control of their enzymatic activities.Our approach is an important addition to the photocaged noncanonical amino-acid toolbox and provides a general method to photocontrol protein activity based on caging a critical glutamate.

A DNA Force Circuit for Exploring Protein-Protein Interactions at the Single-Molecule Level

Protein-protein interactions(PPls)play a crucial role in drug discovery and disease treatment.However,the development of effective drugs targeting PPls remains challenging due to limited methodologies for probing their spatiotemporal anisotropy.Here,we propose a single-molecule approach using a unique force circuit to investigate Ppl dynamics and anisotropy under mechanical forces.Unlike conventional techniques,this approach enables the manipulation and real-time monitoring of individual proteins at specific amino acids with defined geometry,offering insights into molecular mechanisms at the single-molecule level.The DNA force circuit was constructed using click chemistry conjugation methods and genetic code expansion techniques,facilitating orthogonal conjugation between proteins and nucleic acids.The SET domain of the MLL1 protein and the tail of histone H3 were used as a model system to demonstrate the application of the DNA force circuit.With the use of atomic force microscopy and magnetic tweezers,optimized assembly procedures were developed.The DNA force circuit provides an exceptional platform for studying the anisotropy of PPis and holds promise for advancing drug discovery research targeted at PPIs.

Simplified methodology for a modular and genetically expanded protein synthesis in cell-free systems

Genetic code expansion,which enables the site-specific incorporation of unnatural amino acids into proteins,has emerged as a new and powerful tool for protein engineering.Currently,it is mainly utilized inside living cells for a myriad of applications.However,the utilization of this technology in a cell-free,reconstituted platform has several advantages over living systems.The typical limitations to the employment of these systems are the laborious and complex nature of its preparation and utilization.Herein,we describe a simplified method for the preparation of this system from Escherichia coli cells,which is specifically adapted for the expression of the components needed for cell-free genetic code expansion.Besides,we propose and demonstrate a modular approach to its utilization.By this approach,it is possible to prepare and store different extracts,harboring various translational components,and mix and match them as needed for more than four years retaining its high efficiency.We demonstrate this with the simultaneous incorporation of two different unnatural amino acids into a reporter protein.Finally,we demonstrate the advantage of cell-free systems over living cells for the incorporation ofδ-thio-boc-lysine into ubiquitin by using the methanosarcina mazei wild-type pyrrolysyl tRNACUA and tRNA-synthetase pair,which could not be achieved in a living cell.