Development and comparison of cell-free protein synthesis systems derived from typical bacterial chassis

Abstract

Cell-free protein synthesis (CFPS) systems have become an ideal choice for pathway prototyping, protein production, and biosensing, due to their high controllability, tolerance, stability, and ability to produce proteins in a short time. At present, the widely used CFPS systems are mainly based on Escherichia coli strain. Bacillus subtilis, Corynebacterium glutamate, and Vibrio natriegens are potential chassis cells for many biotechnological applications with their respective characteristics. Therefore, to expand the platform of the CFPS systems and options for protein production, four prokaryotes, E. coli, B. subtilis, C. glutamate, and V. natriegens were selected as host organisms to construct the CFPS systems and be compared. Moreover, the process parameters of the CFPS system were optimized, including the codon usage, plasmid synthesis competent cell selection, plasmid concentration, ribosomal binding site (RBS), and CFPS system reagent components. By optimizing and comparing the main influencing factors of different CFPS systems, the systems can be optimized directly for the most influential factors to further improve the protein yield of the systems. In addition, to demonstrate the applicability of the CFPS systems, it was proved that the four CFPS systems all had the potential to produce therapeutic proteins, and they could produce the receptor-binding domain (RBD) protein of SARS-CoV-2 with functional activity. They not only could expand the potential options for in vitro protein production, but also could increase the application range of the system by expanding the cell-free protein synthesis platform.

Characterizing and Improving pET Vectors for Cell-free Expression

“…The presence of N-terminal His and S tags suppressed protein expression in all three CFPS systems to varying extents. Investigating the interplay between N-terminal tags and ribosome binding site (RBS) sequences may also be warranted ( Salis et al, 2009 ; Zhang et al, 2021 ). To evaluate the importance of the RBS, we utilized the extremely useful tool De Novo DNA ( www.denovodna.com ) ( Espah Borujeni et al, 2014 ; Espah Borujeni and Salis, 2016 ; Espah Borujeni et al, 2017 ).…”

Programmable Synthesis of Biobased Materials Using Cell‐Free Systems

“…As an important genetic part for gene expression, RBSs were also tested in CFSs. [ 280–285 ] Rapid quantification of a massive RBS library by liquid handling robot offered an efficient screening method and successfully expanded the available toolset for use with non‐model organisms. [ 278 ]…”

In vivo, in vitro and in silico: an open space for the development of microbe‐based applications of synthetic biology

“…This family of cell‐free systems keeps being improved (Grasemann et al ., 2021). Yet, despite continuous progresses (Zhang et al ., 2021b), sometimes for industrial purposes (at least for the synthesis of proteins used by research laboratories), the speed and accuracy of the translation process remained significantly below what happens in vivo .…”