scholarly journals Using genetically incorporated unnatural amino acids to control protein functions in mammalian cells

2019 ◽  
Vol 63 (2) ◽  
pp. 237-266 ◽  
Author(s):  
Alexander R. Nödling ◽  
Luke A. Spear ◽  
Thomas L. Williams ◽  
Louis Y.P. Luk ◽  
Yu-Hsuan Tsai
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Protein Function ◽  
Unnatural Amino Acids ◽  
Mammalian Cells ◽  
Structural Investigations ◽  
Cellular Translation ◽  
Protein Functions ◽  
Canonical Amino Acid ◽  
Genetic Code Expansion

Abstract Genetic code expansion allows unnatural (non-canonical) amino acid incorporation into proteins of interest by repurposing the cellular translation machinery. The development of this technique has enabled site-specific incorporation of many structurally and chemically diverse amino acids, facilitating a plethora of applications, including protein imaging, engineering, mechanistic and structural investigations, and functional regulation. Particularly, genetic code expansion provides great tools to study mammalian proteins, of which dysregulations often have important implications in health. In recent years, a series of methods has been developed to modulate protein function through genetically incorporated unnatural amino acids. In this review, we will first discuss the basic concept of genetic code expansion and give an up-to-date list of amino acids that can be incorporated into proteins in mammalian cells. We then focus on the use of unnatural amino acids to activate, inhibit, or reversibly modulate protein function by translational, optical or chemical control. The features of each approach will also be highlighted.

scholarly journals Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency

Amino Acids ◽  
2020 ◽  
Author(s):  
Thomas L. Williams ◽  
Debra J. Iskandar ◽  
Alexander R. Nödling ◽  
Yurong Tan ◽  
Louis Y. P. Luk ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Genetic Code ◽  
Unnatural Amino Acids ◽  
Trna Synthetase ◽  
Unnatural Amino Acid ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Genetic Code Expansion ◽  
Incorporation Efficiency

AbstractGenetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

scholarly journals A Genetically Encoded Isonitrile Lysine for Orthogonal Bioorthogonal Labeling Schemes

Molecules ◽  
2021 ◽  
Vol 26 (16) ◽  
pp. 4988
Author(s):  
Ágnes Szatmári ◽  
Gergely B. Cserép ◽  
Tibor Á. Molnár ◽  
Bianka Söveges ◽  
Adrienn Biró ◽  
...  
Keyword(s):  
Genetic Code ◽  
Mammalian Cells ◽  
Protein Structures ◽  
Cycloaddition Reaction ◽  
Membrane Receptors ◽  
Synthetic Dyes ◽  
Double Labeling ◽  
Click Reactions ◽  
Canonical Amino Acid ◽  
Genetic Code Expansion

Bioorthogonal click-reactions represent ideal means for labeling biomolecules selectively and specifically with suitable small synthetic dyes. Genetic code expansion (GCE) technology enables efficient site-selective installation of bioorthogonal handles onto proteins of interest (POIs). Incorporation of bioorthogonalized non-canonical amino acids is a minimally perturbing means of enabling the study of proteins in their native environment. The growing demand for the multiple modification of POIs has triggered the quest for developing orthogonal bioorthogonal reactions that allow simultaneous modification of biomolecules. The recently reported bioorthogonal [4 + 1] cycloaddition reaction of bulky tetrazines and sterically demanding isonitriles has prompted us to develop a non-canonical amino acid (ncAA) bearing a suitable isonitrile function. Herein we disclose the synthesis and genetic incorporation of this ncAA together with studies aiming at assessing the mutual orthogonality between its reaction with bulky tetrazines and the inverse electron demand Diels–Alder (IEDDA) reaction of bicyclononyne (BCN) and tetrazine. Results showed that the new ncAA, bulky-isonitrile-carbamate-lysine (BICK) is efficiently and specifically incorporated into proteins by genetic code expansion, and despite the slow [4 + 1] cycloaddition, enables the labeling of outer membrane receptors such as insulin receptor (IR) with a membrane-impermeable dye. Furthermore, double labeling of protein structures in live and fixed mammalian cells was achieved using the mutually orthogonal bioorthogonal IEDDA and [4 + 1] cycloaddition reaction pair, by introducing BICK through GCE and BCN through a HaloTag technique.

scholarly journals Simplified Methodology for a Modular and Genetically Expanded Protein Synthesis in Cell-Free Systems

2019 ◽  
Author(s):  
Yonatan Chemla ◽  
Eden Ozer ◽  
Michael Shaferman ◽  
Ben Zaad ◽  
Rambabu Dandela ◽  
...  
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Unnatural Amino Acids ◽  
High Efficiency ◽  
Living Cells ◽  
Trna Synthetase ◽  
Complex Nature ◽  
Reporter Protein ◽  
A Cell ◽  
Genetic Code Expansion

ABSTRACTGenetic 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, utilization of this technology in a cell-free, reconstituted platform has several advantages over living systems. The common 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. In addition, 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 of δ-thio-boc-lysine into ubiquitin by using the methanosarcina mazei wild-type pyrrolysyl tRNACUA and tRNA-synthetase pair, which can not be achieved in a living cell.

scholarly journals Making Sense of “Nonsense” and More: Challenges and Opportunities in the Genetic Code Expansion, in the World of tRNA Modifications

2022 ◽  
Vol 23 (2) ◽  
pp. 938
Author(s):  
Olubodun Michael Lateef ◽  
Michael Olawale Akintubosun ◽  
Olamide Tosin Olaoba ◽  
Sunday Ocholi Samson ◽  
Malgorzata Adamczyk
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Protein Design ◽  
Side Chains ◽  
Vast Number ◽  
Trna Modifications ◽  
Wide Range ◽  
Challenges And Opportunities ◽  
Code Extension ◽  
Genetic Code Expansion

The evolutional development of the RNA translation process that leads to protein synthesis based on naturally occurring amino acids has its continuation via synthetic biology, the so-called rational bioengineering. Genetic code expansion (GCE) explores beyond the natural translational processes to further enhance the structural properties and augment the functionality of a wide range of proteins. Prokaryotic and eukaryotic ribosomal machinery have been proven to accept engineered tRNAs from orthogonal organisms to efficiently incorporate noncanonical amino acids (ncAAs) with rationally designed side chains. These side chains can be reactive or functional groups, which can be extensively utilized in biochemical, biophysical, and cellular studies. Genetic code extension offers the contingency of introducing more than one ncAA into protein through frameshift suppression, multi-site-specific incorporation of ncAAs, thereby increasing the vast number of possible applications. However, different mediating factors reduce the yield and efficiency of ncAA incorporation into synthetic proteins. In this review, we comment on the recent advancements in genetic code expansion to signify the relevance of systems biology in improving ncAA incorporation efficiency. We discuss the emerging impact of tRNA modifications and metabolism in protein design. We also provide examples of the latest successful accomplishments in synthetic protein therapeutics and show how codon expansion has been employed in various scientific and biotechnological applications.

Rational design of a function-based selection method for genetically encoding acylated lysine derivatives

2019 ◽  
Vol 17 (25) ◽  
pp. 6127-6130
Author(s):  
Hui Miao ◽  
Chenguang Yu ◽  
Anzhi Yao ◽  
Weimin Xuan
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Directed Evolution ◽  
Rational Design ◽  
Selection Method ◽  
Genetic Code Expansion

Genetic code expansion depends on the directed evolution of aaRS to recognize non-canonical amino acids. Herein, we reported a function-based method that enables rapidly evolving aaRS for acylated lysine derivatives.

scholarly journals The Role of Orthogonality in Genetic Code Expansion

Life ◽  
2019 ◽  
Vol 9 (3) ◽  
pp. 58 ◽  
Author(s):  
Pol Arranz-Gibert ◽  
Jaymin R. Patel ◽  
Farren J. Isaacs
Keyword(s):  
Gene Expression ◽  
Amino Acids ◽  
Genetic Code ◽  
Cross Reactivity ◽  
Elongation Factors ◽  
Translational Machinery ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Genetic Code Expansion

The genetic code defines how information in the genome is translated into protein. Aside from a handful of isolated exceptions, this code is universal. Researchers have developed techniques to artificially expand the genetic code, repurposing codons and translational machinery to incorporate nonstandard amino acids (nsAAs) into proteins. A key challenge for robust genetic code expansion is orthogonality; the engineered machinery used to introduce nsAAs into proteins must co-exist with native translation and gene expression without cross-reactivity or pleiotropy. The issue of orthogonality manifests at several levels, including those of codons, ribosomes, aminoacyl-tRNA synthetases, tRNAs, and elongation factors. In this concept paper, we describe advances in genome recoding, translational engineering and associated challenges rooted in establishing orthogonality needed to expand the genetic code.

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