Research On Protein Synthesis Materials Based On Cell-free Biosynthesis System

With the advancement of cell-free synthetic biology,research has been conducted in various fields such as biomaterials,metabolic engineering,and drug discovery.Protein-based biomaterials are characterized by stability,low immunity,and biocompatibility,and these advantages have led to various bionic materials being fabricated for biomedical applications.However,protein-based biomaterials generally need to be purified by forming monomers inside cells before they can be assembled in vitro.However,due to the complex intracellular environment of organisms and the limitations of cellular activity,it is not possible to precisely regulate the conditions of protein expression and use all the energy to synthesize homogeneous target proteins.To address these challenges,this study proposes a technique for in situ expression and assembly of target proteins,namely the cell-free protein synthesis system（CFPS）technology.Compared to cellular systems,it offers simpler and faster engineering solutions in an open environment with unprecedented design freedom.Several proteins capable of being used in biomaterials were characterized using this technique.Specific results of the study are as follows:（1）The cell-free protein synthesis system（CFPS）was used as a platform to characterize six dihedral symmetric scaffold proteins capable of forming fibril stacks.Firstly,three different reporter proteins（YFP/sf GFP/mCherry）were fused at the N-terminal end of the six scaffold proteins,and the ability of these six scaffold proteins to form protein aggregates ranging from 1-10μm in the CFPS system was demonstrated by various characterization means such as protein immunoblot analysis,laser confocal microscopy,cryo-electron microscopy and gel filtration chromatography.And these polymers were not affected by the fusion proteins and formed more stable protein aggregates.To demonstrate the effect of the scaffold proteins on the fusion proteins,the reporter proteins were switched to functional xylanase（TFX）,and the linkage of these polymers was shown to increase the TFX specific enzyme activity by 0.1-1 fold by testing the TFX specific enzyme activity.The successful in situ synthesis and self-assembly of these scaffold proteins in CFPS not only saves significant time,but also increases the flexibility to add or remove natural or synthetic components.This flexibility and convenience demonstrates the suitability of the CFPS system for biomaterials development and production.（2）The environment of glycoprotein formation was explored using the cell-free protein synthesis system（CFPS）as a platform.Firstly,plasmids expressing glycan(OPS₃₅₅),receptor protein and oligosaccharyltransferase（CTB-pglL）were added to the CFPS system for expression by one-potting,and it was found that no glycoprotein was formed.CTB and pglL were also expressed separately,and the expression of CTB in the CFPS system was determined.Considering the low but efficient expression of pglL in cells and the unknown expression of glycans in CFPS,both were transferred into the host separately and together for expression of glycoproteins in the form of extracts.The expression of glycoproteins was determined in small amounts in the CFPS system with pglL and OPS₃₅₅ added as extracts,respectively.This was followed by optimization and exploration of the CFPS system as well as the extract preparation process.The results did not achieve the same amount of glycoprotein expression as in cells,but have great potential for glycoprotein-based biomaterials synthesis as a simpler and faster glycoprotein synthesis system after continuous optimization and exploration.