Expression of TAT Recombinant Oct4, Sox2, Lin28, and Nanog Proteins from Baculovirus-infected Sf9 Insect Cells

Overview

Journal Gene

Specialty Molecular Biology

Date 2014 Dec 6

PMID 25476026

Citations 7

Authors

Chuanying Pan

Wenchao Jia

Baisong Lu

Colin E Bishop

Affiliations

Soon will be listed here.

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.

Citing Articles

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References

Woltjen K, Michael I, Mohseni P, Desai R, Mileikovsky M, Hamalainen R . piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature. 2009; 458(7239):766-70. PMC: 3758996. DOI: 10.1038/nature07863. View

Takahashi K, Yamanaka S . Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006; 126(4):663-76. DOI: 10.1016/j.cell.2006.07.024. View

Li X, Zhang P, Wei C, Zhang Y . Generation of pluripotent stem cells via protein transduction. Int J Dev Biol. 2014; 58(1):21-7. DOI: 10.1387/ijdb.140007XL. View

Stadtfeld M, Nagaya M, Utikal J, Weir G, Hochedlinger K . Induced pluripotent stem cells generated without viral integration. Science. 2008; 322(5903):945-9. PMC: 3987909. DOI: 10.1126/science.1162494. View

Hacker D, Kiseljak D, Rajendra Y, Thurnheer S, Baldi L, Wurm F . Polyethyleneimine-based transient gene expression processes for suspension-adapted HEK-293E and CHO-DG44 cells. Protein Expr Purif. 2013; 92(1):67-76. PMC: 7129890. DOI: 10.1016/j.pep.2013.09.001. View

Nemes C, Varga E, Polgar Z, Klincumhom N, Pirity M, Dinnyes A . Generation of mouse induced pluripotent stem cells by protein transduction. Tissue Eng Part C Methods. 2013; 20(5):383-92. PMC: 4005486. DOI: 10.1089/ten.TEC.2013.0026. View

Okita K, Hong H, Takahashi K, Yamanaka S . Generation of mouse-induced pluripotent stem cells with plasmid vectors. Nat Protoc. 2010; 5(3):418-28. DOI: 10.1038/nprot.2009.231. View

Ban H, Nishishita N, Fusaki N, Tabata T, Saeki K, Shikamura M . Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors. Proc Natl Acad Sci U S A. 2011; 108(34):14234-9. PMC: 3161531. DOI: 10.1073/pnas.1103509108. View

Chen Y, Liu B, Dai Y, Lee C, Chan M, Chen H . A gene delivery system for insect cells mediated by arginine-rich cell-penetrating peptides. Gene. 2011; 493(2):201-10. DOI: 10.1016/j.gene.2011.11.060. View

10.

Shen X, Hacker D, Baldi L, Wurm F . Virus-free transient protein production in Sf9 cells. J Biotechnol. 2013; 171:61-70. DOI: 10.1016/j.jbiotec.2013.11.018. View

11.

Zhang H, Ma Y, Gu J, Liao B, Li J, Wong J . Reprogramming of somatic cells via TAT-mediated protein transduction of recombinant factors. Biomaterials. 2012; 33(20):5047-55. DOI: 10.1016/j.biomaterials.2012.03.061. View

12.

Inoue H, Nagata N, Kurokawa H, Yamanaka S . iPS cells: a game changer for future medicine. EMBO J. 2014; 33(5):409-17. PMC: 3989624. DOI: 10.1002/embj.201387098. View

13.

Imasaki T, Calero G, Cai G, Tsai K, Yamada K, Cardelli F . Architecture of the Mediator head module. Nature. 2011; 475(7355):240-3. PMC: 4109712. DOI: 10.1038/nature10162. View

14.

Pan C, Lu B, Chen H, Bishop C . Reprogramming human fibroblasts using HIV-1 TAT recombinant proteins OCT4, SOX2, KLF4 and c-MYC. Mol Biol Rep. 2009; 37(4):2117-24. PMC: 3772671. DOI: 10.1007/s11033-009-9680-6. View

15.

Yu J, Hu K, Smuga-Otto K, Tian S, Stewart R, Slukvin I . Human induced pluripotent stem cells free of vector and transgene sequences. Science. 2009; 324(5928):797-801. PMC: 2758053. DOI: 10.1126/science.1172482. View

16.

Ogay I, Lihoradova O, Azimova S, Abdukarimov A, Slack J, Lynn D . Transfection of insect cell lines using polyethylenimine. Cytotechnology. 2008; 51(2):89-98. PMC: 3449679. DOI: 10.1007/s10616-006-9022-7. View

17.

Geisse S . Reflections on more than 10 years of TGE approaches. Protein Expr Purif. 2008; 64(2):99-107. DOI: 10.1016/j.pep.2008.10.017. View

18.

Rasmussen S, Choi H, Rosenbaum D, Kobilka T, Thian F, Edwards P . Crystal structure of the human beta2 adrenergic G-protein-coupled receptor. Nature. 2007; 450(7168):383-7. DOI: 10.1038/nature06325. View

19.

Backliwal G, Hildinger M, Hasija V, Wurm F . High-density transfection with HEK-293 cells allows doubling of transient titers and removes need for a priori DNA complex formation with PEI. Biotechnol Bioeng. 2007; 99(3):721-7. DOI: 10.1002/bit.21596. View

20.

Rajendra Y, Kiseljak D, Baldi L, Hacker D, Wurm F . A simple high-yielding process for transient gene expression in CHO cells. J Biotechnol. 2011; 153(1-2):22-6. DOI: 10.1016/j.jbiotec.2011.03.001. View