Cloning Of TRAIL And Optimization For Its Expression Conditions

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in numerous tumor cell lines while sparing normal cells. Once it officially put into use, large quantities of biologically active TRAIL are needed. As has been reported, TRAIL mainly expressed in the form of inclusion body. Whereas, the low efficiency and complicated process of renaturation, which is inevitable for the inclusion body to get functional, obstructs its mass production. Therefore, reducing the inclusion body and increacing the soluble protein will promote the industrial development of TRAIL.In this study, the cDNA sequence of TRAIL (encoding residues 114-281) was amplified by PCR. Then, the genes were cloned into plasmid pET-28a and expressed in E.coli BL21(DE3). Western blot analysis showed specific reaction of the expressed recombinant protein.The response surface methodology (RSM) was adopted to optimize the culture conditions of TRAIL. The production of soluble TRAIL was improved from 146.84 mg/L to 454.85mg/L after optimization. Furthermore, high-density fermentation was performed. Production of soluble TRAIL concentration reached to 3.66 g/L under the fermentation conditions.By sonication and centrifugation, the supernatant was collected and loaded onto chromatography for purification. TRAIL were purified by Ni-affinity chromatography and ion-exchange chromatography combinational. 1.04g/L of purified TRAIL was obtained after purification. Finally, the purified soluble TRAIL was identified, and the results showed that the recombinant protein was in the condition of high purity, high activity and the molecular weight matched well with the theoretical value.