Applications of genetic code expansion in the study of lysine acylation / 中国药科大学学报

@#Lysine acylation is a ubiquitous protein modification that controls various aspects of protein function. However, it can be challenging to decipher the biological function of site-specific acylation modifications in living cells.The recently developed genetic code expansion (GCE) technology has enabled site-specific incorporation of unnatural amino acids (UAAs) that are structurally consistent with the natural acylation modifications in vivo through orthogonal aminoacyl-tRNA synthetase/tRNA pairs, thus facilitating the study of physicochemical properties and biological behaviors of homogeneously acylated proteins.Besides, GCE technology allows for the targeted introduction of UAAs that mimic acylation modifications but cannot be recognized by deacylases, which improves the stability of lysine acylation modification products.Moreover, the insertion of photo-crosslinked UAAs at specific sites of the target protein has been used to elucidate the reciprocal proteome of acylated modified proteins.Based on the introduction of different structural and functional acylation modifications, we described the novel design of GCE technology combined with three types of UAAs, and their application in studying the functional effects of protein acylation modifications on the enzyme activity, protein stability, cellular localization, protein-DNA interactions and protein-protein interactions of target proteins, with a description of the limitations and prospects of GCE technology in studying protein acylation modification.

Genetic incorporation of unnatural amino acids into proteins and its translational application in biomedicine / 中国药科大学学报

@#Proteins in the human body are usually made of 20 natural amino acids.Through different amino acid combinations and isomerization, proteins of diverse functions are built.An emerging genetic code expansion technology can introduce unnatural amino acids into specific sites of target protein, endowing the protein with new biological characteristics including covalently binding with proximal proteins, carrying fluorescence, and mimicking specific protein post-translational modifications.In this paper, based on the structure and function of unnatural amino acids, the applications of different types of unnatural amino acids in regulating protein''s stability, studying protein''s conformation, expression level, and localization, and uncovering heretofore unknown protein-protein interactions were reviewed.Besides, genetic code expansion of unnatural amino acids is anticipated to find broad utilities in biomedicine by bringing new ideas and methods to the design and optimization of biologics.

Construction and application of microbial cell factories for unnatural amino acids / 生物工程学报

Unnatural amino acids are widely used in medicine, pesticide, material, and other industries and the green and efficient synthesis has attracted a lot of attention. In recent years, with the rapid development of synthetic biology, microbial cell factories have become a promising means for biosynthesis of unnatural amino acids. This study reviewed the construction and application of microbial cell factories for unnatural amino acid, including the synthetic pathway reconstruction, design/modification of key enzymes and their coordinated regulation with precursors, blocking of competitive alternative pathways, and construction of cofactor circulation systems. Meanwhile, on the basis of the new principles for designing the microbial cell factories, new biosynthetic pathways adapted to cells and the production environment, as well as new biomanufacturing system established based on cell adaptive evolution and intelligent fermentation regulation, we looked forward to the further construction and application of microbial cell factories for industrial bio-production.

Genetic code expansion and its application in characterization and imaging of proteins / 生物工程学报

Genetic code expansion (GCE) allows the incorporation of unnatural amino acids into proteins via using stop codons. GCE may achieve site-specific labeling of proteins in combination with the click reaction. Compared with other labeling tools such as fluorescent proteins and tagged antibodies, the compound molecules used in protein labeling by GCE technology are smaller, and therefore, may less interfere the conformational structure of proteins. In addition, through click reaction, GCE allows a 1:1 stoichiometric ratio of the target protein molecule and the fluorescent dye, and the protein can be quantified based on the fluorescence intensity. Thus, GCE technology has great advantages in the researches that require the exposition of living cells under high laser power for longer time, for example, in the context of single molecule tracing and super-resolution microscopic imaging. Meanwhile, this technology lays the foundation for improving the accuracy of positioning and molecule counting in the imaging process of living cells. This review summarized the GCE technology and its recent applications in functionally characterizing, labeling and imaging of proteins.

Recent advances in cell-free unnatural protein synthesis / 生物工程学报

Cell-free unnatural protein synthesis (CFUPS) as an emerging approach for protein engineering research, has been successfully applied in basic scientific studies (e.g., protein-protein interaction, protein-nucleic acid interaction) and industrial production (e.g., pharmaceutical proteins, protein materials). CFUPS can improve the engineering freedom and the process control by allowing free addition of genetic elements and chemicals as research purposes. It also can give protein novel characteristics, structures and functions, including post-translational modification of proteins, incorporation of reaction handles, synthesis of biophysical probes and polymeric protein. This article systematically reviews the unnatural amino acid incorporation methods (global suppression, stop codon suppression, frameshift suppression and unnatural base-pairs) and the application advances of unnatural amino acids in protein modifications, biophysical probes, enzyme engineering, biomaterials and biopharmaceutical protein production. The opportunities and challenges of the CFUPS system development and the wide application of industrialization are also illustrated with details.