The Study Of The TAT-mediated Protein Transduction Of The TAT-Rhox5 That Prified From E.Coli

Objective: The penetrating peptides, usually no more that 20 amino acids, are able topenetrate the cellular membrane including the nuclear evelope without damaging it. This naturalor artificial polypeptides, which are water soluble and of low spallation, can also penetratevarious cellular membranes through the inphagocytosis. Amongst them, the Trans-activatingtranscriptional activator of the Human immunodeficiency virus, TAT, was studied intensively.TAT or its derivatives can transport both micromolecule substance and macromolecule drugs, aswell as nanometer substance to difference living mammalian cells. Rhox gene family is a newhomeobox gene cluster which allocates on the X chromosome and expresses only in female andmale mice reproduction system. Despite its potential role in regulating the embryonicdevelopment, particularly the development of germinal tissue and the production and maturity ofsperm, the cellular function of Rhox5 was left largely unknown. In previous study, wedemonstrated interactions between Rhox5 and mPem,. Cdc37 and Mdfic in a yeast two-hybridscreen of a 7-day mouse embryo library. In this study, a TAT-Rhox5 fusion protein wasexpressesed and purified in E.Coli for further functional study of Rhox5.Method: The sequence encoding TAT was cloned into pET22b(+)-Rhox5 in frame of Rhox5.The resulted pET22b(+)-TAT-Rhox5 was transformed into Rosseta^XM2(DE3). The expression ofTAT-Rhox5-6His fusion protein was induced by IPTG and verified by Western blot assay. TheTAT-Rhox5 was purified and used to transduct COS-7 cells subsequently. The intracellularlocalization of TAT-Rhox5 was observed by immumofluorescence.Result: The pET22b(+)-TAT-Rhox5 prokaryotic expression vector was constructedsuccessfully and the TAT-Rhox5-6His fusion protein was highly expressed in E. coil. Thepurified TAT-Rhox5 fusion protein was uptaken by NIH3T3 cells. Except a small amount innucleus, TAT-Rhox5 showed a cytoplasmic distribution in the tested cells. The best contration oftransduce of TAT-Rhox5 fusion protein is 3μM. When the transduce time was increase, there aremore fusion protein distribution in nucleus.Conclusion: The TAT-Rhox5 fusion protein was purified successfully and it was uptaken byNIH3T3 cells. TAT-Rhox5 fusion protein was distribution in cytoplasmic and nucleus, the resultwas settling a foundation for further functional study of Rhox5.