Construction,expression and intracellular localization of truncation mutants of Max interacting protein 1-0

Objective: To construct the recombinant eukaryote expression vectors of Max interacting protein 1-0(Mxi1-0) truncation mutants and detect the intracellular localization of the mutants.Methods: Five kinds of truncation mutants were generated by polymerase chain reaction with a template of enhanced green fluorescent protein eukaryotic expression vector(pEGFP-N1) containing Mxi1-0 gene.Subsequently,the target sequences were subcloned into pEGFP-N1.The recombinant vectors were confirmed by restriction enzyme digestion and DNA sequencing.The mutants were transfected into mouse embryo fibroblast(NIH3T3).Fusion protein in NIH3T3 cells were detected by Western blot.The intracellular localization of mutants was investigated by immunofluorescence.Results: The recombinant eukaryote expression vectors of Mxi1-0 mutants were constructed successfully.The expression of these mutants in NIH3T3 cells could be detected by Western blot.In addition,the immunofluorescence results showed that in NIH3T3 cells,proline rich domain(PRD) mutant was in cytosol and nuclei,putative Sin3-interacting domain(tSID) mutant was mainly in cytosol,little in nuclei.PRD-tSID mutant and △PRD mutant were mainly in cytosol,while △PRD-tSID mutant was mainly in nuclei.Conclusions: The truncation mutants of Mxi1-0 were constructed and expressed successfully in NIH3T3 cells,the localization in cells was also observed,which will be benefit for the future research on the mechanism of Mxi1-0 localization and function.