Screening,expression And Purification Of PEP-1-MIT-hSOD2 K29R Mutant And Its Function On Anti-parkinson's Disease In Vitro

Purpose: This study fused mutant human manganese superoxide dismutase?hSOD2?and protein transduction domain PEP-1 and then studied the protective effects of mitochondrial targeting proteins PEP-1-MIT-hSOD2 mutant on MPP⁺ induced SH-SY5 Y cell.Methods: Bioinformatic softwares?SYBYL and Clustal X?and online forecasting platforms?Swiss-model and HOTMUSIC?were used to predict eighteen mutants of hSOD2.The mutants were constructed using p ET-15b-hSOD2 as a template.Then the PCR products were transformed into E.coli BL21.After induced with the same conditions,paraquat?PQ?resistance was used to screen the hSOD2 mutants with high SOD activity.Inductively coupled plasma mass spectrometry?ICPMS?was used to analyze the Mn²⁺ binding ability of mutant SOD2.A commercial kit was used to analyze the SOD activity of the mutants after treatment with different concentrations SDS,different p H and different temperature for 1 h.Then a recombination expression vector PEP-1-MIT-hSOD2 K29 R mutant was constructed.E.coli BL21 was used as host cells to express the recombination protein PEP-1-MIT-hSOD2 mutant.After purified with Ni-NTA,immunofluorescence was used to analyze the location.The protective effect of PEP-1-MIT-hSOD2 K29 R on MPP⁺ exposed SH-SY5 Y cells were detected by MTT [3-?4,5-dimethyl-2-thiazolyl?-2,5-diphenyl-2-H-tetrazolium bromide].The DCF-DA?2'7'-dichlorofluorescein diacetate?probe was used to detect the effect of PEP-1-MIT-hSOD2 K29 R on generation of ROS in MPP⁺ induced SH-SY5 Y cells.Real-time PCR was used to detect the effect of PEP-1-MIT-hSOD2 K29 R on expression level of inflammatory factor and apoptosis factor.Results:?1?Eighteen hSOD2 mutant recombination expression vectors were constructed in this study.hSOD2 K29 R with a higher SOD activity compared with the wild type hSOD2 was screened.?2?Three-dimensional modeling found that the distance between Mn²⁺ and N1 atom of H74 in activity center changed from 2.05 ? to 2.01 ?.ICPMS data showed that Mn²⁺ binding ability of hSOD2 K29R was 0.966±0.025,increased around 1.05-fold compared with wild type hSOD2.The SOD activity of the mustant hSOD2 K29R was higher than wild type hSOD2 after treatment with different concentration SDS,different p H and different temperature for 1 h.?3?The recombinant expression vector p ET-28a-PEP-1-MIT-hSOD2 K29 R was constructed.The per liter of LB medium can express 59 mg PEP-1-MIT-hSOD2 K29 R proteinat most.?4?PEP-1-MIT-hSOD2 K29 R can increase the viability of MPP⁺ exposed SH-SY5 Y cells from 53.4% to 81.6%.PEP-1-MIT-hSOD2 K29 R has no cytotoxicity to SH-SY5 Y cells.?5?PEP-1-MIT-hSOD2 K29 R protein can locate into mitochondria of SH-SY5 Y cells and reduce the level of ROS induced by MPP⁺.1 ?M recombinant protein PEP-1-MIT-hSOD2 K29 R could down-regulate the expression of inflammatory factor TNF-? and IL-6 and increase the proportion of Bcl-2/Bax in MPP⁺ induced SH-SY5 Y cells.Conclusion: The mutant hSOD2 K29R with higher SOD activity compared with wild type hSOD2 was got in this study.The recombinant protein PEP-1-MIT-hSOD2 K29 R can locate into mitochondria.The recombinant protein PEP-1-MIT-hSOD2 K29 R could down-regulate the expression of inflammatory factor TNF-?,IL-6 and increase the ratio of Bcl-2/Bax.The fusion protein PEP-1-MIT-hSOD2 K29 R could develop a new medicine for PD.