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, 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-thioboc-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.

Emerging Methods for Efficient and Extensive Incorporation of Non-canonical Amino Acids Using Cell-Free Systems

“…Recently, cell extracts of RF1-deficient strains were also utilized in a CFPS system for efficient and modular artificial protein expression with multiple different ncAAs, but partial competition was still present, similar to the cells ( Hong et al, 2014 ; Ozer et al, 2017 ; Chemla et al, 2019 ). Further, several total competition-removing CFPS methods were developed using different strategies.…”

Emerging Methods for Efficient and Extensive Incorporation of Non Canonical Amino Acids using Cell-free Systems

“…Recently, cell extracts of RF1-deficient strains were also utilized in a CFPS system for efficient and modular artificial protein expression with multiple different ncAAs, but partial competition was still present, similar to the cells [48,84,85]. Further, several total competition-removing CFPS methods were developed using different strategies.…”

Exploring protein phosphorylation by combining computational approaches and biochemical methods

“…Despite the evident strengths of CFPS, a great variability in reaction performance has also been documented, the causes of which can be multiple (such as ncAA sequence location in the protein construct) and unmanageable [84] . Some authors have developed strategies to obtain pSer, pThr, and pTyr by adding a protective “hood” against the action of phosphatases [73] .…”