title: Expression of TAT recombinant Oct4, Sox2, Lin28, and Nanog proteins from baculovirus-infected Sf9 insect cells.
abstract: Somatic cell reprogramming has generated enormous interest, following the first report of generation of induced pluripotent stem cells (iPSCs) from mouse fibroblasts, but the integration of viral transgenes into the genome is unlikely to be accepted. Given these safety considerations, a method for virus-free transient gene expression from suspension-adapted Sf9 insect cells was developed. Here, we expressed transactivator of transcription (TAT)-fused proteins, Sox2, Oct4, Lin28, and Nanog in Sf9 cells using the baculovirus expression vector system (BEVS). The molecular weights of the TAT-Sox2, TAT-Oct4, TAT-Lin28, and TAT-Nanog fusion proteins were 36kD, 40kD, 24kD, and 36kD, respectively. Further investigation indicated that most of the recombinant proteins remained in the nuclei of the Sf9 insect cells and were therefore unavailable for purification and cellular reprogramming. Once this problem has been solved, it seems likely that protein expressed from baculovirus-infected Sf9 insect cells will be the method of choice for cellular reprogramming.
authors.affiliation: College of Animal Science and Technology, Shaanxi Key Laboratory of Molecular Biology for Agriculture, Northwest A&F University, Yangling, Shaanxi 712100, PR China; Institute for Regenerative Medicine, Wake Forest University, 391 Technology Way, Winston-Salem, NC 27101, USA.
authors.forename: Chuanying
authors.initials: C
authors.lastname: Pan
authors.affiliation: College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, PR China.
authors.forename: Wenchao
authors.initials: W
authors.lastname: Jia
authors.affiliation: Institute for Regenerative Medicine, Wake Forest University, 391 Technology Way, Winston-Salem, NC 27101, USA.
authors.forename: Baisong
authors.initials: B
authors.lastname: Lu
authors.affiliation: Institute for Regenerative Medicine, Wake Forest University, 391 Technology Way, Winston-Salem, NC 27101, USA.
authors.forename: Colin E
authors.initials: CE
authors.lastname: Bishop
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D065150:Cellular Reprogramming; D018398:Homeodomain Proteins / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism*; D055748:SOXB1 Transcription Factors / Q000378:metabolism; D061987:Sf9 Cells; D018411:Spodoptera / Q000821:virology*; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000378:metabolism
chemical_list: D018398:Homeodomain Proteins; D050814:Octamer Transcription Factor-3; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D055748:SOXB1 Transcription Factors; D054322:tat Gene Products, Human Immunodeficiency Virus
keywords: Gene expression; Induced pluripotent stem cells (iPSCs); Recombinant protein; Sf9 insect cells