Expression, purification, and refolding of active Nrf2 transcription factor fused to protein transduction TAT tag.
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor responsible for activation of diverse genes that protect the cell against xenobiotics and oxidative stress. The Nrf2-dependent transcription is tightly controlled by cytoplasmic interaction of Nrf2 with its inhibitor, Kelch ECH-associating protein 1 (Keap1). The Keap1-mediated inhibition can be overcome by addition of xenobiotics or by overexpression of Nrf2 protein. The overexpressed Nrf2 overwhelms the Keap1 inhibition, translocates into the nucleus and activates antioxidant response element (ARE)-dependent gene transcription that protects the cell against oxidative stress. We expressed and purified recombinant mouse Nrf2 protein fused to a protein transduction domain (TAT), derived from transactivator of transcription protein from HIV-1. Full-length TAT-Nrf2 was expressed in Escherichia coli in insoluble inclusion bodies and purified to homogeneity using denaturing size exclusion chromatography. Optimal refolding conditions were determined through the use of a light scattering-based refolding assay and analytical size exclusion chromatography. The results demonstrate that the refolded TAT-Nrf2 could transduce into cultured human neuroblastoma cells. The transduced TAT-Nrf2 activated transcription of ARE-dependent genes and conferred protection against intracellular reactive oxygen species (ROS) generated by hydrogen peroxide exposure.
Chumanov
Robert S
RS
McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI 53706, USA.
Burgess
Richard R
RR
D000818:Animals; D045744:Cell Line, Tumor; D015696:Gene Products, tat; D006801:Humans; D006861:Hydrogen Peroxide; D051267:NF-E2-Related Factor 2 / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D018384:Oxidative Stress; D015202:Protein Engineering; D017510:Protein Folding
D015696:Gene Products, tat; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D006861:Hydrogen Peroxide