Purification And Biological Activity Measure Of The Transcription Factor’s Recombinant Proteins

Oct4and Sox2are the main regulators maintaining self-renewal and multipledifferentiation potential ability, and are crucial to reprogram somatic cells to inducedpluripotent stem cells (iPSCs). The newest research orientation is to preparerecombinant proteins of transcription factors mOct4and mSox2for induced iPSCssafely instead of DNA transfection. The solubility and purity of recombinant proteins isso low that it reduces efficiency and quality of iPSCs. Expression and purification ofrecombinant proteins is essential to ensure protein entry into cells and act astranscription factors efficiently.The codons of transcription factors Oct4and Sox2were optimized in theexperiments. The recombinant proteins were expressed by expression system of BL21（DE3）and pET. Multiple-step purification and cationization increased the purity andconcentration. Then the recombinant proteins were marked by fluorescent reagent toobserve motion. The stability of proteins in cells was measured used Western Blot. Thevector with luciferase reporter gene and target gene Nanog regulation sequence wasconstructed to identify the protein’s transcriptional activity with stable cell line andrecombinant proteins. The results show that the concentration and the purity of twoproduced proteins prepared could be improved. The proteins in the medium could enterthrough cells spontaneously within1-2hours and existed stably above36hours byWestern Blot. Target gene Nanog was effectively activated by recombinant proteinsmOct4and mSox2. Bone marrow mesenchymal stem cells had great potential ability ofmulti-directional differentiation and reproductive activity with recombinant proteinsmOct4. The proteins prepared were high solubility, high purity, and high activity toincrease induced productivity of iPSCs. The basis of efficient technique to induce iPSCswas established in the experiment.