Material-assisted Optimization Of Cell-free Protein Expression

Following the rapid development in the field of biotechnology,the use of synthetic biology has emerged as a quick and efficient tool for building complex life systems.In order to overcome the inherent limitation of synthetic biology by living cells,anew research field,cell-free protein synthesis system,is proposed as a powerful and flexible technology that manipulate biological systems for life science applications.In particular,cell-free systems have been widely employed in the platform technology development of industrialized high-throughput protein production.In order to achieve high yield expression of cell-free systems;one guiding principle is to simulate the cytoplasmic environment in cell-free system to form the macromolecular crowding effect.In this paper,we propose to combine the characteristics of materials – nanoclay,thermosensitive hydrogel,metal organic framework – with gene templates to simulate the crowded environment of macromolecule in a cell-free system,ultimately to improve upon protein expression yield.As an inorganic-organic hybrid functional nano-materials,nanoclay forms a hydrogel with electrostatic attraction after mixing with an ionic solution.The adsorption effect immobilizes the plasmid on its surface,thereby forming a macromolecular crowded environment in the cell-free system.The use of it resulted in an increase the protein production of cell-free systems by 1.75 times.Thermosensitive hydrogel has the property of inducing a volume phase change at an ambient temperature above 34 °C.By utilizing this property,the plasmid encapsulated inside the hydrogel can be released and aggregate on the surface of the hydrogel as the temperature rises,successfully forming a crowded environment of macromolecules.The results show that it can increase the protein production of cell-free systems by 3.7 times.Metal organic framework(MOF)has the property of encapsulating biomolecules in a stable manner.This feature allows the plasmid to be encapsulated and form a compartment in cell-free system to form a macromolecular crowded environment.The results show 2.7 times increase in protein yield.In addition,the kinetics of these three materials participating in cell-free system was analyzed,and it was found that they they synthesize proteins more quickly and efficiently than the original acellular system.Finally,the transcription level of the materials involved in cell-free reaction is significantly improved,improving the enzyme conversion efficiency of cell-free reaction.This paper confirms that the macromolecular crowded environment created by materials can increase the concentration of gene template,and achieve high protein expression in cell-free system,which provides technical support for the subsequent development of industrialized production platform of cell-free system.