In vitro suppression of two different stop codons

Abstract

Proteins play a crucial role in all living organisms, with the 20 natural amino acids as their building blocks. Unnatural amino acids are synthetic derivatives of these natural building blocks. These amino acids have unique chemical or physical properties as a result of their specific side chain residues. Their incorporation into proteins through ribosomal translation in response to one of the stop codons has opened a new way to manipulate and study proteins by enabling new functionalities, thus expending the genetic code. Different unnatural amino acids have different functionalities, hence, the ability to incorporate two different unnatural amino acids, in response to two different stop codons into one protein is a useful tool in protein manipulation. This ability has been achieved previously only in in vivo translational systems, however, with limited functionality. Herein, we report the incorporation of two different unnatural amino acids in response to two different stop codons into one protein, utilizing a cell-free protein synthesis system. Biotechnol. Bioeng. 2017;114: 1065-1073. © 2016 Wiley Periodicals, Inc.

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

“…One of the most widely used protocols for extract preparation is the S30 protocol [13,18,24,[27][28][29][30][31][32], which was modified in this study to create the genetically expanded cell-free protein synthesis system. When using orthogonal translation systems (OTS) to achieve GCE harbored on a replicating bacterial vector, such as the pEVOL [33] vector, the extract preparation protocol becomes much longer and more difficult as the bacterial growth becomes slower.…”

“…Recently, we have found that extracts from the same bacterial strain but with different genotypes and from different batches could be mixed and combined [13], this led us to develop a modular approach for cell-free protein synthesis with GCE. In this approach, a component of choice, which could be a protein or a non-protein-coding gene like tRNAs, could be expressed separately in bacteria which will sequentially be prepared as an extract that contains the said The results clearly show that this approach is viable for at least four different combinations of interacting components of tRNAs and tRNA synthetases.…”

“…[34]. The buffer amount per reaction and the expression plasmid concentration were performed as described before [13]. All plasmids were purified from E. coli DH5a cells using the Wizard Plus SV minipreps kit [Promega].…”

“…Another critical advantage for genetically expanded cell-free protein synthesis is the absence of the host genome, which removes limitations of toxicity and off-target suppression that may occur by suppression of the host endogenous stop codons. This is even more pronounced when two different Uaas are utilized at the same time; an exploit that is possible in living cells but is simplified in cell-free platforms [13].…”

“…Lastly, we will promote a modular approach that was recently introduced [13], where different endogenous components could be expressed in separate bacterial extracts, stored for long periods and combined in a desired combinatorial fashion when needed [ Fig 1]. Figure 1 | General scheme of the simplified preparation protocol and the modular approach for the cell-free reaction presented in this study.…”