Construction of 3*Flag-PAK4 recombinant plasmid and its protein expression and localization in COS7 cell line

Objective: To construct the expression plasmid of 3*Flag-hPAK4 and identify its recombinant protein expression and localization,and purify the Pak4 protein.Methods: Total RNA was extracted from human breast cancer MCF-7 cells.The hPAK4 coding sequence was amplified by polymerase chain reaction(PCR) method and subcloned into 3*Flag vector.After the target region was sequenced,the plasmid was transfected into COS7 cell line.The expression of the recombinant plasmid in COS7 cells was proved by Western blotting.The localization of 3*Flag-hPAK4 in COS7 cells was observed by using laser scanning confocal microscopy.The hPak4 protein was purify by immunoprecipitate.Results: hPAK4 had been constructed into expressing vector 3*Flag successfully.The length of the fragment was 1 800 bp,identified by restriction enzymes digestion.The expression of 3*Flag-hPAK4 fusion protein was detected by Western blot,with a molecular weight 68 kDa,was pulled down by Flag antibody,its localization in the cytoplasm.Conclusion:The recombinant plasmid is successfully cloned into eukaryotic expressing vector,the expression of 3*Flag-hPAK4 fusion protein is identified and pulled down by Flag antibody,it was expressed in cytoplasm.