Prokaryotic And Eukaryotic Fusion Expression Of TAT-reprogramming Factors

Induced pluripotent stem cells(iPS) were generated to resemble embryonic stem cells(ES). Owing to their abilities to replicate indefinitely and to differentiate into numerous cell types, and non-ethical constraints and no rejection risk of their application, generation of iPS was thought to be one of the most important breakthroughs in the field of Cellular Biology and Regenerative Medicine. Previous studies found that some proteins those with protein transduction domain(PTD) can penetrate the cell membrane and get into the cells. Both Trans-Activator of Transcription(TAT) which was derived from the human immunodeficiency virus type 1(HIV1) and poly arginine ammonia acid(9R) can facilitates cell penetration of the recombinant proteins.This study intended to construct pTAT-bOct4-9R、pTAT-bSox2-9R、pTAT-bKlf4-9R and pTAT-bcMyc-9R reprogramming factors recombinant vectors and express fusion proteis in prokaryotic cells. At the same time, by the help of Baculovirus Expression Vector System(BEVS), pBAC-3/TAT-Oct4 、 pBAC-3/TAT-Sox2 、 pBAC-3/TAT-Lin28 and pBAC-3/TAT-Nanog recombinant vectors were constructed and fusion proteis were then expressed in Sf9 cells. We therefore proposed to test the hypothesis that recombinant reprogramming proteins expressed in Escherichia coli and Sf9 insect cells, carrying the TAT cell-penetrating motif or 9R, would be able to enter somatic cells and reprogram them without involvement of virus. If this can be achieved, it will be the method of choice for reprogramming in domestic animals, such as bovine. 1. Construction and expression of pTAT-bOct4-9R、pTAT-bSox2-9R、pTAT-bKlf4-9R and pTAT-bcMyc-9R prokaryotic recombinant vectorsBased on the sequence of bovine Oct4、Sox2、Klf4 and c Myc gene coding regio n in NCBI, the primers containing restriction sites and 9R(added at the C-terminus) were designed. The PCR products were then sub-cloned into expression plasmid pTATHA and varifiated by enzyme digestion and sequencing. The varificated recombinant expression vectors were named as pTAT-bOct4-9R、pTAT-bSox2-9R、pTAT-bKlf4-9R and pTAT-bcMyc-9R, respectively.After transformated these recombinant plasmids into Escherichia coli BL21(DE3)PLySs cells, expression of fusion proteins were induced at different concentrations of IPTG, temperature and time. The expression products were analysised by SDS-PAGE and Western blot. The optimal induction conditions of pTAT-bSox2-9R、pTAT-bKlf4-9R and pTAT-bc Myc-9R were 0.6 mmol/L IPTG inducing at 37℃ for 4 hours, and the m olecular weights of these three protein were 36 kDa、52.6 kDa and 50 kDa, respective ly. The research results accumulated raw data for the PTD mediated reprogramming fa ctors protein directly programming of bovine somatic cells to generate induced pluripot ent stem cells. 2. Construction and expression of pBAC-3/TAT-Oct4 、 pBAC-3/TAT-Sox2 、pBAC-3/TAT-Lin28 and pBAC-3/TAT-Nanog eukaryotic recombinant vectorsBased on the sequence of human Oct4、Sox2、Lin28 and Nanog gene coding region in NCBI, the primers containing restriction sites and TAT(added at the N-terminus) were designed. The PCR products were then sub-cloned into plasmid pCR4-TOPO and varifiated by enzyme digestion and sequencing. After sequence verification, the target fragments were restricted with EcoRI/SmaI/SrfI andNot I, followed by sub-cloning into the equivalent sites of the baculovirus transfer plasmid, pBAC-3. The varificated recombinant expression vectors were referred as pBAC-3/TAT-Oct4、pBAC-3/TAT-Sox2、pBAC-3/TAT-Lin28 and pBAC-3/TAT-Nanog, respectively.Expression constructs in the pBAC3/TAT-gene baculovirus transfer plasmid were transfected into Sf9 insect cells, and Sf9 cells were then infected with recombinant virus 5 days after transfection. The expression of fusion proteins were analysised by SDS-PAGE and Western blot 72 h after infection. The molecular weights of these fusion proteins TAT-Sox2, TAT-Oct4, TAT-Lin28 and TAT-Nanog were 36 kDa, 40 kDa, 24 kDa and 36 kDa, respectively. Further analysis of protein purification showed that most of the recombinant protein was located in the nucleus, but not in the cytoplasm. This is not conducive to the protein purification and target cells reprogramming.