Development of a cell-free protein synthesis system for practical use

Abstract

Conventional cell-free protein synthesis systems had been the major platform to study the mechanism behind translating genetic information into proteins, as proven in the central dogma of molecular biology. Albeit being powerful research tools, most of the in vitro methods at the time failed to produce enough protein for practical use. Tremendous efforts were being made to overcome the limitations of in vitro translation systems, though mostly with limited success. While great knowledge was accumulated on the translation mechanism and ribosome structure, researchers rationalized that it may be impossible to fully reconstitute such a complex molecular process in a test tube. This review will examine how we have solved the difficulties holding back progress. Our newly developed cell-free protein synthesis system is based on wheat embryos and has many excellent characteristics, in addition to its high translation activity and robustness. Combined with other novel elementary technologies, we have established cell-free protein synthesis systems for practical use in research and applied sciences.

Cell-Size Space Regulates the Behavior of Confined Polymers: From Nano- and Micromaterials Science to Biology

“…The effect of such a membrane surface has also been observed in the transcription–translation (TX–TL) system. A cell-free protein synthesis (CFPS) system, in which the equipment of the transcription–translation system in living cells was prepared in a test tube, , was used to demonstrate this point. The CFPS system reconstitutes the flow of genetic information in which DNA information is transcribed to mRNA (transcription) and then decoded to proteins (translation).…”

“…The acceleration of CFPS activity in the cell-sized space is observed only in CFPS systems using RRL, and we did not observe a similar effect in CFPS systems using other TX−TL equipment. Since previous studies have shown that RRL contains some translation inhibitors that interact with the membrane, 90 the inhibitory factor attached to the membrane loses the inhibitory function, thus accelerating CFPS activity in the cell-sized space. In any case, these experimental results show that the localization of molecules changes in the cell-size space, and this localization shift affects the activity of biochemical systems such as TX−TL.…”

Characterization and Utilization of Disulfide-Bonded SARS-CoV-2 Receptor Binding Domain of Spike Protein Synthesized by Wheat Germ Cell-Free Production System

“…Cell-free protein expression systems offer the advantage of a simple and rapid procedure to produce good-quality proteins, retaining their biologically relevant quaternary structure [ 13 ]. One of the most well-known systems of this type is the wheat germ cell-free protein expression system (WG) [ 14 ]. However, the problem with conventional WG is the use of dithiothreitol (DTT) in the wheat germ extract and substrate solution to enhance translation efficiency [ 15 ].…”

In Vitro Synthesis and Design of Kinesin Biomolecular Motors by Cell-Free Protein Synthesis

“…In contrast to in vivo protein production, cell-free protein synthesis (CFPS) is a rapid and easy method to produce proteins without involving living cells and is used in many research areas. , The reconstituted biomolecular transcription and translation (TXTL) of genes allows the synthesis of a wide variety of desired functional proteins from a DNA template, either a plasmid or a PCR product possessing the gene encoding the target protein. To synthesize proteins using CFPS, specific facilities are unnecessary except for an incubator to control the temperature for the TXTL reaction.…”

“…We chose the wheat germ cell-free system to synthesize kinesin-1 and its variants because it has very low levels of endogenous protease and nuclease activity and provides high levels of expression of a wide range of proteins with high solubility. , The gene encoding GFP-kinesin-1 (k560GFP), which is truncated at 560 amino acids and tagged with GFP, was cloned downstream from the 5′-untranslated region (5′-UTR) consisting of the SP6 promoter and E01 translation enhancer sequence in the pEU-vector (Figure A). GFP-kinesin-1 was translated from the mRNA transcript synthesized by in vitro transcription using the plasmid template.…”