Efficient Soluble Expression And Preliminary Study On Extracellular Secretion Of STNFR1 In Escherichia Coli

The massive release of tumor necrosis factor(TNF-?)in vivo can mediate the occurrence of many diseases.It mainly exerts its physiological activity through binding to receptors on the membrane(type ? and type ?).Soluble tumor necrosis factor type ? receptor(sTNFR1)is the extracellular domain of tumor necrosis type ? receptor(TNFR1).It can competitively block the binding of TNFR1 to TNF and reduce the toxicity of TNF on cells.Therefore,sTNFR1 can be used as an antagonist of TNF-?,and combined with TNF-? for the treatment of diseases,has important research value in the medical field.In this study,the heterologous expression of sTNFR1 in Escherichia coli was achieved by optimizing the expression system,and obtain higher purity of sTNFR1 protein,which laid a good foundation for studying the physiological activity of sTNFR1,the application of disease treatment,and its large-scale expression and preparation as a pharmaceutical protein in the future.In this study,the human sTNFR1 gene was cloned into the pET-22 b expression vector and the recombinant plasmid pETH1 was successfully constructed.The pETH1 was transformed into E.coli BL21(DE3)host strain for shake flask fermentation.The result showed that the all target protein was expressed in the form of inclusion bodies.In order to increase the soluble expression of sTNFR1 in E.coli,firstly,fusion tag technology was used,we select four kinds of fusion tags including DsbC,MBP,TrxA and NusA,.The results showed that compared with the other three tags,fusion of the NusA tag at the N-terminus of sTNFR1 had the best effect,which increased the soluble expression of sTNFR1 by 20%.Secondly,in order to further promote the soluble expression of sTNFR1,on the basis of NusA-sTNFR1,seven intracellular chaperone plasmids or thiol oxidase Erv1 in yeast disulfide de novo synthesis system were co-expressed to assist the correct folding of sTNFR1 protein.The expression of sTNFR1 protein was significantly promoted by tig chaperones,and the soluble expression of sTNFR1 in E.coli was up to 90%.The EC12 strain was shake flask fermentation,the fermentation conditions were optimized from the induction temperature and IPTG induction concentration.The results showed that the induction temperature was 25 oC,the final concentration of IPTG was 0.5 mM,and TB was used as fermentation culture.At the base,the total and intracellular soluble expression levels of sTNFR1 protein were the best.Ni-NTA affinity chromatography was used to purify the soluble protein,the NusA tag was removed by enzymatic hydrolysis with TEV protease,Combined with Western Blot analysis,it was confirmed that the expressed product was sTNFR1 protein.The results of previous studies were further optimized the expression system to study extracellular secretion of sTNFR1 protein in E.coli.First,the appropriate signal peptides were screened to construct the strains with the five signal peptides of OmpA,LamB,DsbA,TorT and phoA.The results showed that the signal peptide TorT could induce sTNFR1 secretion,but Low secretion.In order to increase the extracellular secretion of sTNFR1,the secretion bottleneck may be analyzed because the target protein cannot cross the cell membrane and the secretion of the protein is blocked.Therefore,phospholipase C and cutinase are co-expressed to increase the permeability of the cell membrane,thereby promoting protein secretion.The results showed that there was no significant increase in extracellular secretion,and further analysis of its secretion bottleneck was needed.