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Fusion Expression and Penetrating Ability Study of Cell Penetrating Peptide TAT and Induced Pluripotent Stem Cell Related Transcriptional Factors

Author: WangJun
Tutor: JinJian
School: Jiangnan University
Course: Microbial and Biochemical Pharmacy
Keywords: TAT iPS inclusion body purification
Type: Master's thesis
Year: 2013
Downloads: 42
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The generation of induced pluripotent stem cell (iPS) has become the main focus of stemcell research since somatic cells were successfully reprogrammed into stem like cells usingectopic expression of different reprogramming factors: Oct4, Sox2, Klf4, c-Myc. However, theinsertion of exogenous genes into genome increases the risk of tumorigenicity, which mayhave an effect on the clinical application of iPS technology. Therefore, protein-based methodprovides an alternative and ideal technology for generating therapeutical iPS. In this study, weexploited the cell penetrating ability of TAT to obstain TAT-Oct4, TAT-Sox2, TAT-Klf4,TAT-c-Myc and TAT-Nanog fusion proteins as a basis for generating safer iPS.TAT-Oct4, TAT-Sox2and TAT-Klf4target genes were amplified using PCR technology.TAT-c-Myc and TAT-Nanog were amplifed using overlapping PCR. All PCR products weredigested with Nco I and Xho I and then inserted into the pET28a expression vector. Therecombinant expression plasmids were verified by sequencing. Then the recombinant plasmidwas transformed into BL21(DE3) expression strain. The SDS-PAGE analysis showed thatrecombinant strains respectively habouring TAT-Oct4, TAT-Sox2, TAT-Klf4, TAT-c-Myc andTAT-Nanog were obstained.The target proteins TAT-Oct4, TAT-Sox2, TAT-Klf4, TAT-c-Myc and TAT-Nanog weremainly expressed with insoluble inclusion bodies in E.coli. And after inclusion bodieswashing and Ni sepharose purification, the purity of each target protein was more than90%.The addition of chemical additives such as arginine, glycerol and GSH/GSSG during ureagradient dialysis improved the average refolding yield of each protein which was8.8%,51.0%,47.9%,67.2%,8.5%respectively.Immunocytochemistry analysis showed TAT-mediated iPS related factors couldefficiently penetrate target cells. And the optimal concentration for penetrating cells was6μg/mL,8μg/mL,8μg/mL,8μg/mL and4μg/mL individually. In order to detect the relativequantity of each protein in cells, Flow cytometry analysis showed the fluorescence intensityvalue of each protein was37.5,44.5,38.7,44.7,64.7and the relative fluorescence intensityratio was16.6%,20.6%,17.3%,20.7%and33.2%.

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