【Abstract】 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 blot. The localization of 3*Flag-hPAK4 in COS7 cells was observed by using laser scanning confocal microscopy. Purify the hPak4 protein by immunoprecipitate. Results hPAK4 had been constructed into expressing vector 3*Flag successfully. The length of the fragment was 1800bp, identified by restriction enzymes digestion. The expression of 3*Flag-hPAK4 fusion protein was detected by Western blot, with a molecular weight 68KDa, was pulled down by Flag antibody, its localization in the cytoplasm. Conclusion The recombinant plasmid was successfully cloned into eukaryotic expressing vector, the expression of 3*Flag-hPAK4 fusion protein was identified and pulled down by Flag antibody, was expressed in cytoplasm.