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

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.

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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 ◽

10.1007/s00726-020-02927-z ◽

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.

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Expanding the Genetic Code of Yeast for Incorporation of Diverse Unnatural Amino Acids via a Pyrrolysyl-tRNA Synthetase/tRNA Pair

Journal of the American Chemical Society ◽

10.1021/ja104609m ◽

2010 ◽

Vol 132 (42) ◽

pp. 14819-14824 ◽

Cited By ~ 128

Author(s):

Susan M. Hancock ◽

Rajendra Uprety ◽

Alexander Deiters ◽

Jason W. Chin

Keyword(s):

Amino Acids ◽

Genetic Code ◽

Unnatural Amino Acids ◽

Trna Synthetase

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Genetic Code Expansion of Mammalian Cells with Unnatural Amino Acids

Current Protocols in Chemical Biology ◽

10.1002/9780470559277.ch150038 ◽

2015 ◽

Vol 7 (3) ◽

pp. 187-199

Author(s):

Kalyn A. Brown ◽

Alexander Deiters

Keyword(s):

Amino Acids ◽

Genetic Code ◽

Unnatural Amino Acids ◽

Mammalian Cells ◽

Genetic Code Expansion

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

Essays in Biochemistry ◽

10.1042/ebc20180042 ◽

2019 ◽

Vol 63 (2) ◽

pp. 237-266 ◽

Cited By ~ 19

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.

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Pyrrolysyl-tRNA-Synthetase: Methanogenese und Gencode-Erweiterung

BIOspektrum ◽

10.1007/s12268-021-1653-x ◽

2021 ◽

Vol 27 (6) ◽

pp. 616-619

Author(s):

Nikolaj Georg Koch ◽

Nediljko Budisa

Keyword(s):

Amino Acids ◽

Genetic Code ◽

Methanogenic Archaea ◽

Trna Synthetase ◽

Side Chains ◽

Site Specific ◽

Genetic Code Expansion ◽

The Way

AbstractPyrrolysyl-tRNA synthetase (PylRS) is an enzyme of some methanogenic Archaea for the natural incorporation of pyrrolysine into proteins. The discovery of PylRS as a natural tool for genetic code expansion paved the way for site-specific incorporation of non-canonical amino acids (ncAAs) into proteins, with versatile side chains useful in biotechnology. Almost 20 years after the discovery, we describe the journey which led to three distinct classes of PylRSs with unique ncAA recognitions.

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Photoreactive Bicyclic Amino Acids as Substrates for MutantEscherichia coliPhenylalanyl-tRNA Synthetases

Journal of Biological Chemistry ◽

10.1074/jbc.m401278200 ◽

2004 ◽

Vol 279 (19) ◽

pp. 19839-19845 ◽

Cited By ~ 20

Author(s):

Thomas Bentin ◽

Ramin Hamzavi ◽

Johan Salomonsson ◽

Hervé Roy ◽

Michael Ibba ◽

...

Keyword(s):

Amino Acids ◽

Protein Engineering ◽

Unnatural Amino Acids ◽

High Efficiency ◽

Trna Synthetase ◽

Double Ring ◽

Reactive Groups ◽

Model Protein

Unnatural amino acids carrying reactive groups that can be selectively activated under non-invasive biologically benign conditions are of interest in protein engineering as biological tools for the analysis of protein-protein and protein-nucleic acids interactions. The double ring system phenylalanine analogues benzofuranylalanine and benzotriazolylalanine were synthesized, and their photolability was tested by UV irradiation at 254, 320, and 365 nm. Although both showed photo reactivity, benzofuranylalanine appeared as the most promising compound because this amino acid was activated by UVA (long wavelength) irradiation. These amino acids were also tested forin vitrocharging of tRNAPheand for protein mutagenesis via the phenylalanyl-tRNA synthetase variant αA294G that is able to facilitatein vivoprotein synthesis using a range ofpara-substituted phenylalanine analogues. The results demonstrate that benzofuranylalanine, but not benzotriazolylalanine, is a substrate for phenylalanine tRNA synthetase αA294G, and matrix-assisted laser desorption ionization time-of-flight analysis showed it to be incorporated into a model protein with high efficiency. Thein vivoincorporation into a target protein of a bicyclic phenylalanine analogue, as described here, demonstrates the applicability of phenylalanine tRNA synthetase variants in expanding the scope of protein engineering.

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