Research On The Construction And Optimization Of Cell-free Systems

With the development of biotechnology,cell-free protein synthesis technology has made it possible to inexpensively express and recombinant proteins rapidly.Many highly active CFPS platforms are now available and have recently been used in synthetic biology.The use of cell-free protein synthesis system enables the rapid expression of functional proteins using genes.It plays an important role in display technology,non-natural amino acid insertion and in vitro protein evolution,and provides a platform for studying protein translation and folding.Among the cell-free systems,E.coli cell-free system has the advantages of low cost,high yield,and easy preparation,making it the most widely used cell-free system.Firstly,based on the cell-free synthesis system of E.coli extract,t His-Tag paper optimizes the expression of fluorescent protein,pharmaceutical protein and complex protein under different conditions,and found the cell extract species,magnesium ion concentration and redox environment,the degree of crowding of the reaction system and the gene sequence affect the expression of the protein in the system,and it is the starting point for exploring the expression system of different proteins.In order to provide a basis for the construction of a portable protein production platform,t His-Tag paper explored the stability of the cell-free system in the form of lyophilized powder,and tested the fluorescent protein de GFP as a signal protein at room temperature,30?,and 37?.We tested the stability of all-component-freeze-dried and lyophilized in group,and tried to propose a better storage strategy,it was found that the lyophilization of the group can better retain the activity of each part,but as for the storage strategy,The form of the whole component lyophilization is better than the form in which the fractions are lyophilized and mixed,higher protein expression levels can be obtained.In addition,we successfully constructed and expressed 34 proteins necessary for transcription and translation,and confirmed their expression in vivo in order to test the role of each protein in the synthesis process.We attempted to set up the PURE system more easily by using these proteins,and the purification step was simplified by group purification.As a result,the system thus prepared was found to be inactive and required further optimization.