Recombinant Expression And Bioactivity Analysis Of TRAIL-Fc Fusion Proteins With Different Linkers In E.coli

Similar to TNF-α and Fas-L, TRAIL is one of the members of TNF family, and hasbroader anticancer spectrum than the formers. TRAIL can combine with the death receptorand induce apoptosis of tumor cells without obvious effect or severe inflammatoryresponse on normal cells, which has been using to develop anti-cancer drugs.However, theshort half-life in vivo has greatly limited its effects. Recombinant construction of chimerictarget-IgG1Fc proteins was employed to increase the half-life of recombinant protein drug,which could also reduce the potential liver toxicity of TRAIL. Such protocol is an idealmethod to enhance the safety and half-life of TRAIL protein.Objective:In this study, E.coli secretory expression system was used to construct and expressseries TRAIL-Fc proteins containing different linkers. Candidate TRAIL-Fc proteins wereselected based on the flask expression and characterization of the stability of linkers.Homogeneous Trail-Fc proteins were obtained after screening high-level expressed strainsand optimizing fermentation conditions and purification process. Supervisor TRAIL-Fcstructure was confirmed in the basis of structure confirmation and in vitro and in vivobioability evaluation, which may guide the further development of anti-cancer drugs.Methods:①Design and construct recombinant expression plasmid pET-22b-TRAIL-Fc withdifferent Linkers, and transforme into host strain E.coli BL21(DE3) plysS by CaCl2.②Induced the expression of recombinant TRAIL-Fc by IPTG. Detecte and screen thestrain which could express protein with stable structure and not easy to fracture inHinge region. Ferment in30L culture medium.③Optimize the purification process of TRAIL-Fc protein.Purified the target protein by Qion exchange chromatography column,protein A affinity chromatography column andCM ion-exchange chromatography column.Then,study on physicochemical propertiesof protein.④Detecte the inhibitory effect of TRAIL-Fc on multiple strains of tumor cell.Establish aH22mouse model of liver cancer to detecte the inhibitory effect on tumor cell in vivo.Result: ①The cDNA of TRAIL-Fc-L1、TRAIL-Fc-L2、TRAIL-Fc-L3and TRAIL-Fc-L4werecloned into pET-22b and the plasmid pET-22b-TRAIL-Fc-L1、pET-22b-TRAIL-Fc-L2、pET-22b-TRAIL-Fc-L3and pET-22b-TRAIL-Fc-L4was transformed into E.coliBL21(DE3)plysS successfully.②The results detected by SDS-PAGE electrophoresis and liquid chromatography-massspectrometry LC-MS assay showed that the structure of recombinant TRAIL-FC-L3and TRAIL-FC-L4is more stable and the hinge region did not breakage.③The stable purification procedure was established and the degree of purity was up to90%. The results of SDS-PAGE、mass spectrometry and western blot showed that therecombinant protein TRAIL-Fc-L3、TRAIL-Fc-L4had expected molecular weight andimmunogenicity.④Result of activity experiment in vitro showed that the inhibitory effect of TRAIL-Fc-L3protein is more obvious to various tumor cell, and TRAIL-Fc-L3is sensitive to thetumor cells which TRAIL-sensitive not. Result of activity experiment in vitro showedthat TRAIL-Fc-L3could inhibit the growth of tumor cells significantly and no sideeffects toliver toxic of mouse,while the half-life of TRAIL-Fc increased significantly invivo and played a very good effect with once every three days.