Abstract:

To construct the GST-tagged eukaryotic expression vectors of human p21-activated kinase 1(hPAK1) autoinhibitory domain(AID) with different activities and identify the expressions of their recombinant proteins,and to explore the function of PAK1 AID and molecular targeting role in tumor therapy.Methods The pcDNA3.1HisC-PAK1 full length plasmid was used as template,and the fragment of wild PAK1 AID was amplified with PCR，and the fragment of PAK1 L107F was amplified with long primer mutant method.The PCR fragments were double-digested and cloned into pEBG.The wild and inactive types of pEBG-PAK1 were transfected into HEK293 cells and their expressions were identified by Western blotting.Results The wild and in active PAK1 AID  fragments with 200 bp  were obtained by PCR.5 000 bp vector band and 200 bp PAK1 AID band were obtained by double-digestion.The wild and inactive types of  pEBG-PAK1  were expressed in HEK293 cells,and the molecular weight of the protein was 33 000.Conclusion The wild and inactive types of PAK1 AID  eukaryotic expression vectors are successfully constructed.The expressions of wild and inactive  types of PAK1 AID proteins  are identified.

Key words: p21-activated kinase 1, autoinhibitory domain, inactive type（L107F）, eukaryotic expression, GST-tag