The Expression Of Recombinant Protein Of PAK1 And Its Location In Cells

ObjectivePAK1 ( P21 - activated kinase 1) is an evolutionarily conserved serine/threonine kinases, are becoming increasingly important for a variety of cellular functions in mammalian cells. Pak1 acts as downstream effectors for the small GTPas-es, Cdc42 and Racl. Pak1 is highly expressed in the brain, muscle, and spleen. PAK1 contains an N - terminal regulatory domain and a C - terminal kinase domain, its N -terminal regulatory domain contains GTPase binding domain to mediate the binding of PAK1 to Rac/Cdc42. Accumulating evidence indicates that PAK1 is important for a variety of cellular functions including cell morphogenesis, motility, survival, mitosis, cell cycle and angiogenesis. Pak1 are involved in the regulation of cellular function via phosphorylating a number of downstream target protein. Many evidences showed that Pak1 activation occurs during the process of tumorigenesis, and Pak1 is likely to provide insight into the role of Paks in human cancers. In this study,I detect PAK1 expression in pro-karyocyte and its location in transfected eukaiyocyte so as to provide basis for further study of PAK1.MethodsTotal RNA was isolated using Tripure agent. The cDNA of all kinds of tissues were prepared using M - MuLV Reverse Transcriptase and Oligo dT primer.Techniques of gene recombination and gene transfection were employed.The coding sequences of PAK1 were cloned into the procaryotic expression vector pGEX - 5X - 1 , generating the recombinant plasmids pGEX - 5X - 1/ PAK1, expressed in BL21 after transformation and IPTG induction. In the first stage of a Western Blot, SDS -PAGE electrophoresis was used to separate proteins, and the proteins was seperated on the gel based on size. In the second stage of the Western Blot, Proteins from the gel were transferred onto a membrane using electrophoresis. The proteins was transferred to the membrane in the same position as they are on the gel. Finally, I used anti - GST SRP and PAK1 antibodies to detect the GST -PAK1 fusion protein.At the same time,PAK1 cDNA was ligated to EGFP gene of pEGFP - C1. The recombinant plasmid was transfected into cancer cell line with liposome, and the expression of GFP - PAK1 fusion protein was observed by fluorescent microscopy .ResultsAfter isolation of total RNA, the samples were quantitated on the basis of absorbance at 260/280nm. Their concentration and 260/280 value could meet the need of its application.When the recombinant plasmid was detected with double endonulease analysis , two fragments could be released from it. And their size is correspondent to vector and target gene respectively. Moreover, sequence analysis indicated that PAK coding sequence was completely correct. These evidences showed that PAK1 recombinant plasmid was successfully constructed.GST - PAK1 fusion protein could be expressed in 1 ,2,4 hrs after IPTG induction and reached its peak at 2hr. Western blot analysis indicated that the fusion protein could specifically bind to anti - GST and PAK1 antibody. So we may determine that PAK1 recombinant protein could be expressed in prokaryo-cyte.Under fluorescent microscopy, cells which highly expressed GFP protein showed green fluorescence. The expression of PAK1 was observed in cytoplasm transfected with pEGFP - PAK1.