The Transmembrane Fusion Protein Of MnSOD Clone Expression And Function Study

Superoxide dismutase (SOD) is important purger eliminate O2- out of body, it can prevent and protect the destruction and damage of O2- to human body,and has anti-inflammatory,anti-radiation and anti-tumor effects.SOD as a biological macromolecules,it difficult to cross the cell membrane and play a biological effect.So we choose protein transduction domain of TAT and R9,connect its encoding gene with MnSOD by PCR,then constructed recombinant plasmids and expression the fusion protein in E.coli;at last we compare the transmembrane,anti-tumor and anti-radiation ability of fusion protein Mn-SOD. Mn-SOD-TAT,Mn-SOD-R9 and Mn-SOD-EC-S. It has three parts:First of all, we successfully synthetic the primers contains protein transduction domain TAT and R9, plasmids pET24a-MnSOD as a template,PCR amplification the fusion gene includes Mn-SOD-TAT and Mn-SOD-R9; then we constructed recombinant expression plasmids pET28aMn-SOD-TAT and pET28aMn-SOD-R9;the recombinant expression plasmids transformed into E.coli BL21(DE3), IPTG induce fusion protein in 37℃,the result of total bacterial is nearly 40%,mainly in the form of insoluble inclusion bodies.Then, we optimize the fusion protein expression condition through change the inducer temperature,the concentration of manganses ions and IPTG and research the purification of fusion protein.MnSOD-TAT optimai expression condition is 0.2mmol/L IPTG and 0.6mmol/L Mn2+ in 28℃,the inducer time is 12 hours, then the activity of supernatant soluble can get 500U/ml. MnSOD-R9 optimai expression condition is 0.42mmol/L IPTG and 0.6mmol/L Mn2+ in 28℃,the inducer time is 12 hours, then the activity of supernatant soluble can get 700U/ml.Under normal conditions,soluble fusion protein can not have a good combination with Ni-NTA,we deal with this problem by adding 2M urea in sample to purify,the protein purify is higher than 90%.Finally, we verify the recombinant proteins transmembrane ability through FITC-protein;MTT assay the effect of recombinant proteins to cancer cell growth and protect from UV radiation and H2O2 injury. Mn-SOD-TAT,Mn-SOD-R9 and Mn-SOD-EC-S can go into the cell more easily than Mn-SOD,they can play a important role in inhibit cancer cell grow and protect cell from UV radiation and H2O2 injury.Transmembrane and other biological effects is concentration and time related.Different protein transduction domain have their own advantages in anti-radiation, anti- H2O2 injury and anti-tumor effects.