Optimization Of Cell-free Protein Synthesis System And Its Utilazation On Proinsulin Expression In Liposome

Objective To investigate the effect of transcription-translation components on cell-free synthesis(CFPS)system.And to optimaze the CFPS system.Utilize the CFPS and liposome to build a CFPS-liposome system for proinsulin expression,So that to lay a foundation for further development of an oral insulin intestinal transport system.Methods Molecular cloning technology was used in plasmids construction.Recombinant proteins were overexpressed in Escherichia coli and were purified using Ni-NTA affinity column.BCA protein assay kit determined the protein concentration.Preparation of the cell extract for CFPS system utiliazed Escherichia coli.Protein synthesis substrates and energy were added to construct CFPS system and enhanced fluorescence protein(EGFP)as a model protein were synthesized by adding corresponding plasmid.The effects of transcription-translation components on CFPS system were investigated by titration.The profitable ones were selected and combined to further optimaze the CFPS system,and super folding fluorescent protein(sfGFP)and proinsulin-EGFP were chosen to test the versatility.Channel protein a-hemolysin(a-HL)were obtained through plasmid constrution,protein expression and purification method.Used membrane staining method and confocal microscope to compare the methods of liposome preparation and chose the best one to construct a CFPS-liposome system for proinsulin expression.Pore forming protein a-HL were introduced to compare the expression difference of CFPS-liposome system with and without channels.Results 30 transcription-translation components were successfully expressed and purified,including 3 initiation factors(IF1,IF2,IF3),3 elongation factors(EF-G,EF-Tu,EF-Ts),2 release factors(RF2,RF3),ribosome recycle factor(RRF),18 aminoacyl-tRNA synthetases(ARS),methionyl-tRNA folmytransferase(MTF),RNA helicase and T7 RNA polymerase(T7 RNAP).And the titration result showed that,among these enzymes and factors,only T7 RNAP,aspartate-tRNA synthetase(AspRS),cysteine-tRNA synthetase(CysRS),IF2,IF3,EF-Tu,EF-Ts,EF-G,RF2,RF3 and RRF could improve CFPS system while IF1 has opposite effect.Supplementary of T7 RNAP,RRF,IF2,IF3 and EF-Tu to CFPS system at the same time increased the synthesizd EGFP to 2.5 fold.This optimized method also increased sfGFP production to 3 fold and proinsulin-EGFP to more than 2 fold,confirming the versatility of optimized CFPS system.a-HL was successfully expressed and purified.Water-in-oil emulsion transfer method was the best one to prepare liposome and was used to construct the CFPS-liposome system.Proinsulin-EGFP and proinsulin-mcherry were expressed by CFPS-liposome and the introduction of α-HL enhanced the expressions.Conclusion CFPS system was optimized by supplementary of T7 RNAP,RRF,IF2,IF3 and EF-Tu at the same time.A CFPS-liposome system was constructed by encapsulating the CFPS system into liposome,and proinsulin were synthesized under the protection of lipid bilayer.