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

Abstract

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 of δ-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.

Cell-Free Biocatalysis for the Production of Platform Chemicals

“…The current literature is also replete with top-down and bottom up suggestions on the way forward for CFS in the synthesis of biocommodities (for example, Lu, 2017;Arbige et al, 2019;Bowie et al, 2020;Libicher et al, 2020). There are many valuable reviews that summarize the status of multi-enzyme arrays and cascades and their possible immobilization all mention the perceived advantages of CFS (for example, Katzen et al, 2005;Smith et al, 2014;Guo et al, 2017;Sperl and Sieber, 2018;Chemla et al, 2019;Hwang and Lee, 2019;Tinafar et al, 2019;Bowie et al, 2020;Giannakopoulou et al, 2020;Silverman et al, 2020). In all cases, considerable faith has been put in the prospect of present and future genetic manipulations to improve the performance of various proteins.…”

Codon-Reduced Protein Synthesis With Manipulating tRNA Components in Cell-Free System

“…Looking forward, this emphasized tRNA complement with protein synthesis system has the potential to be applied in various emerging fields, such as the construction of minimal cells (Noireaux et al, 2011;Stano and Luisi, 2013;Yue et al, 2019), the employment of microfluidic devices (Damiati et al, 2018;Weiss et al, 2018), the incorporation of multiple different ncAAs (Zheng et al, 2018;Chemla et al, 2019;Italia et al, 2019), the direct preparation of bio-conjugates (Bundy et al, 2008;Zimmerman et al, 2014) and other novel biotechnology applications. Aiming at controllably reconstituting a minimum set of compounds, the simplified genetic codon with the CFPS system will play a significant role in the bottom-up assembly of minimal cells.…”

Crystal Structure of Pyrrolysyl-tRNA Synthetase from a Methanogenic Archaeon ISO4-G1 and Its Structure-Based Engineering for Highly-Productive Cell-Free Genetic Code Expansion with Non-Canonical Amino Acids

“…The PylRS·tRNA Pyl pairs are useful for non-canonical amino acid incorporation because of their “orthogonality” (non-reactivity) to the 20 canonical aaRS·tRNA pairs in many organisms [ 15 , 16 , 17 , 18 , 19 ]. PylRS and its mutants showed broad specificity for substrate amino acids, and by using the PylRS·tRNA Pyl pairs, site-specific incorporations of more than 200 non-canonical amino acids into proteins have been achieved in bacteria including Escherichia coli , and eukaryotes including Saccharomyces cerevisiae , mammalian cells, and multicellular organisms (reviewed in [ 10 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 34 ]), and by cell-free protein synthesis based on an E. coli cell extract [ 28 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 ]. Cell-free protein synthesis systems, which are novel protein expression platforms, are particularly suitable for synthesizing cell-toxic proteins and transmembrane proteins that are difficult to synthesize in cellular systems, and can efficiently introduce non-canonical amino acids into such proteins for pharmaceutical research.…”