Production Of Soluble TRAIL Using A Novel Self-cleavable Tag System Fh8-ΔI-CM

Escherichia coli is an essential host for large-scale expression of heterologous polypeptides.However,further applications are limited by the formation of potential protein aggregates.Therefore,there is an ever-increasing need for economical,simple and effective method for protein soluble expression and purification.In this work,we studied the soluble expression of recombinant TRAIL in E.coli.First,we adopt Fh8 tag fusion with TRAIL,Fh8-TRAIL fusion protein was successfully expressed in soluble form,the soluble expression achieved 67.3%among the ’total protein.We conducted HIC in parallel with IMAC for the first purification process.After the first purification step,the fusion protein was cleaved by tobacco etch virus(TEV)protease to release TRAIL.Another purification step was then conducted,and we developed a dual protein purification protocols.In particular after the first HIC process and tag removal were followed with a further IMAC process,or after the first IMAC process and tag removal were followed with a further HIC process.The purity and yield of released TRAIL achieved 97.8%and 69.4 mg respectively when use HIC followed with IMAC process.We then employed a self-cleavage intein to replace TEV protease in an inexpensive,simple Fh8-AI-CM system.Fh8-AI-CM-TRAIL fusion protein was successfully expressed in soluble form,the soluble expression achieved 91.2%among the total protein.After optimized the cleavage efficiency we use a on column purification method by HIC to released TRAIL and the purity achieved 86.4%.After a further IMAC purification,the final yield and purity achieved 82.1 mg/L and 98.4%Respectively.Western bolt indicating that all the purified TRAIL from these two method can conbine to the anti-TRAIL antibody.Besides,In vitro activity demonstrated that the released TRAIL could induce the apoptosis of NCI-H460 but not in normal FBHE cell.Finally,we optimized the fermentation conditions of Fh8-ΔI-CM-TRAIL fusion proteins.The influences of different medium,culture and expression conditions on the soluble expression of fusion protein were evaluated by a single factor experiment.The most three obvious influence fators was investigated by orthogonal test.Finally,the production of recombinant TRAIL increased from 82.1 mg/L to 90.2 mg/L.