TI - Expression and Detection of Biological Active Affinity S-tagged Rex-1 . AB - To identify the PHOSphorylated amino acid residues of Rex-1 , we employed a tandem affinity purification approach of Rex-1 that was over expressed in mammalian cells . The S-tagged Rex-1 vector ( S-Rex-1 ) expressed full-length Rex-1 protein fused to amino terminal His6 and S-tags (Fig 1B) . Since the HTLV-1 regulatory proteins Tax and Rex are expressed from the same mRNA in partially overlapping reading frames , a point mutation was made in the nucleotide sequence that added a stop codon in the tax-1 reading frame that left the Rex-1 amino acid sequence unchanged [44] . This mutation completely abrogated Tax-1 protein expression and function ( data not shown ) . The S-Rex-1 expression construct was transiently transfected into 293T cells , and the appropriate nuclear subcellular localization of Rex-1 was confirmed by indirect immunofluorescence microscopy ( Fig 1C ) . Wild type Rex-1 was shown as a single 27 kDa band by Western blot analysis using rabbit polyclonal alpha-Rex-1 antisera ( Fig 1D ) . Next we determined if the S-tagged Rex-1 retained its ability to function in our quantitative reporter bioassay in which HIV-1 p24 Gag production is measured and used as a read-out of Rex-1 functional activity in cultured cells . It is important to note that this assay has been a proven and accepted assay for Rex function and has been shown to directly correlate to Rex activity in the context of a molecular clone [25,28,45] . As shown in Figure 2A , S-Rex-1 displayed significant functional activity , although slightly lower than wtRex-1 . We hypothesize that this reduced activity likely is due to the proximity of the amino terminal tag to the RNA binding domain . Taken together , these data demonstrate the proper nuclear subcellular localization and efficient expression of a functionally active S-tagged Rex-1 from mammalian cells .