Expression, Purification And Preliminary Application Of PTD-GFP And PTD-EDAG Fusion Proteins

EDAG is a nuclear protein which plays important roles in the regulation of hematopoietic proliferation, differentiation and apoptosis. But the mechanism of it is still unknown. So it is of great significance to transduce EDAG protein into hematopoietic stem cells to study its effect on the regulation of hematopoietic proliferation, differentiation and apoptosis.A novel protein transduction technique PTD (protein transduction domain) was employed to efficiently deliver EDAG protein into hematopoietic stem cells. GFP protein was utilized to systematically optimize the processes of the protein transduction technique. Four PTD-GFP plasmids, including VP22-GFP, Tat-GFP, Tat-GFP-Tat and Tat-GFP-9R, were constructed using recombinant DNA technology. Then fusion proteins expressed in E. coli BL21 were purified by Ni-NTA purification system. To obtain the optimal outcome of each fusion protein, we compared expression conditions such as inducer concentration, the OD and induction time. The native conditions and hybrid conditions were also used to distinguish whether PTD fusion protein purified under hybrid conditions has stronger transmembrane ability as previously postulated. The results showed that fusion proteins purified under Hybrid conditions exhibited stronger cell penetration ability and Tat-GFP-Tat protein possessed the highest protein transduction ability.Then other four PTD-EDAG plasmids, including VP22-EDAG, Tat-EDAG, Tat-EDAG-Tat and Tat-EDAG-9R were constructed and expressed in E. coli BL21. All the proteins were successfully purified and validated using Western-Blot except VP22-EDAG because of its low expression quantity.Tat-EDAG-Tat fusion protein was finally obtained after constructing several PTD-EDAG plasmid vectors and systematically optimizing the expression, purification and transduction processes.