Cell-free Protein Synthesis System Of Pichia Pastoris And Escherichia Coli

Cell-free protein synthesis（CFPS）system is an ideal platform for rapid and convenient protein research.CFPS system has the characteristics of no cell membrane and no cytotoxicity,and all energy can be used to produce target protein.Based on these advantages,a variety of cell-free systems have been developed for different fields of bioengineering,such as macromolecular assembly,unnatural amino acid embedding,glycoprotein production,and so on.At present,the prokaryotic cell-free transcription and translation mainly uses wild-type Escherichia coli as the chassis,but the extract components of wild-type E.coli are complex,which may potentially interfere with the target reaction;at the same time,its own complex metabolic reaction may compete with its engineering reaction in cell-free system in terms of substance and energy,which limits its application effect.On the other hand,most eukaryotic cell-free protein synthesis systems are expensive and time-consuming,so they cannot be produced in large quantities.To optimize the cell-free transcription-translation system based on the crude extracts of prokaryotes and eukaryotes,we used genome-reduced E.coli and Pichia pastoris as host organisms to construct CFPS systems.（1）The CFPS system of P.pastoris was established,and the protein yield was nearly doubled by adding RNase inhibitors.According to the changing trend of protein yield,it was determined that the protein yield could be maximized in 6 hours.The optimum addition volume of cell extract was 47%（v/v）.In order to provide sufficient energy for the continuous reaction,the optimum energy concentrations（40mM CP and 0.8 mg/mL CPK）are determined.Then,the optimization of metal ions which could affect the assembly and activity of ribosomes showed that 80 m M Mg²⁺and 8 m M K⁺could achieve the best synthesis yield.（2）The functional application of P.pastoris CFPS system was explored,and cricket paralysis virus（CRPV）was selected from 14 kinds of IRES as the best IRES translation initiation element.Kozak sequence was constructed into the expression plasmid,which successfully achieved better protein synthesis and successfully achieved the expression of h ACE2-615 protein.（3）The CFPS system of genome-reduced E.coli was established,and the incorporation of single and multiple unnatural amino acids in the system was explored.Among them,△W3110 showed excellent synthetic yield for the 23-site TAA incorporation of p Pa F.In this study,two cell-free transcriptional translation systems were successfully constructed and optimized.In the P.pastoris system,CRPV sequences and Kozak sequences that can promote translation were identified,and h ACE2-615 protein expression was achieved.In the E.coli system,embedding of single and multisite UNAAs was achieved.This experiment can be followed up by using both cell-free systems for biosensing and other studies to expand their applications.