Purification of full-length human Pregnane and Xenobiotic Receptor: polyclonal antibody preparation for immunological characterization

Abstract

Pregnane and Xenobiotic Receptor (PXR; or Steroid and Xenobiotic Receptor, SXR), a new member of the nuclear receptor superfamily, is thought to modulate a network of genes that are involved in xenobiotic metabolism and elimination. To further explore the role of PXR in body's homeostatic mechanisms, we for the first time, report successful prokaryotic expression and purification of full-length PXR and preparation of polyclonal antibody against the whole protein. The full-length cDNA encoding a 434 amino acids protein was sub-cloned into prokaryotic expression vector, pET-30b and transformed into E. coli BL21(DE3) cells for efficient over expression. The inclusion body fraction, containing the expressed recombinant protein, was purified first by solubilizing in sarcosine extraction buffer and then by affinity column chromatography using Ni-NTA His-Bind matrix. The efficacy of anti-PXR antibody was confirmed by immunocytology, Western blot analysis, EMSA and immunohistochemistry. The antibody obtained was capable of detecting human and mouse PXR with high specificity and sensitivity. Immunofluorescence staining of COS-1 cells transfected with human or mouse PXR showed a clear nuclear localization. Results from immunohistochemistry showed that level of PXR in liver sections is immunologically detectable in the nuclei. Similar to exogenously transfected PXR, Western blot analysis of cell extract from HepG2 and COLO320DM cells revealed a major protein band for endogenous PXR having the expected molecular weight of 50 kDa. Relevance of other immunodetectable bands with reference to PXR isoforms and current testimony are evaluated. Advantages of antibody raised against full-length PXR protein for functional characterization of receptor is discussed and its application for clinical purposes is envisaged.

A Phosphomimetic Mutation at Threonine-57 Abolishes Transactivation Activity and Alters Nuclear Localization Pattern of Human Pregnane X Receptor

“…After blocking with Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE) for 1 h, membranes were incubated for 1 h with primary antibodies against FLAG-tag (mouse monoclonal anti-FLAG M2 antibody; Sigma-Aldrich) at 1:1000 dilution or silencing mediator of retinoid and thyroid receptor (SMRT) (mouse monoclonal anti-SMRT antibody; GeneTex, Irvine, CA) at 1:500 dilution. In p70 S6K overexpression experiments, hPXR was probed using rabbit polyclonal anti-hPXR serum (Saradhi et al, 2005). Infrared dye-conjugated secondary goat anti-rabbit (LI-COR Biosciences) or goat anti-mouse (LI-COR Biosciences) antibodies were diluted to 1:20,000 and then used to incubate the membrane for 1 h. The sites of antibody-antigen reaction were visualized using an Odyssey infrared imager (LI-COR Biosciences).…”

“…The cells were blocked in Odyssey blocking buffer (LI-COR Biosciences) for 2 h and then incubated with primary antibody in the blocking buffer for 2 h, followed by four 5-min washes with PBS containing 0.1% Tween 20. Cy3-conjugated anti-FLAG M2 mouse monoclonal antibody (Sigma-Aldrich), rabbit polyclonal anti-hPXR serum (Saradhi et al, 2005), and mouse monoclonal anti-SMRT antibody (GeneTex) were used at 1:200, 1:500, and 1:100 dilutions, respectively. After incubating with appropriate secondary antibody in the blocking buffer for 1 h, the cells were washed with PBS containing 0.1% Tween 20 for a total of four washes with 5 min/wash.…”

“…Oligonucleotide competition was performed using a 500-fold molar excess of unlabeled oligonucleotides. To validate the specific binding between FLAG-hPXR and hot oligonucleotide, 2 g of either mouse monoclonal anti-FLAG M2 antibody (Sigma-Aldrich) or rabbit polyclonal anti-hPXR serum (Saradhi et al, 2005) was added to some reactions. To verify the specific binding between FLAGhPXR and the antibodies, 2 g of either normal mouse IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or normal rabbit IgG (Santa Cruz Biotechnology, Inc.) was added to some reactions as negative controls.…”

Protein Phosphatase 2Cβl Regulates Human Pregnane X Receptor-Mediated CYP3A4 Gene Expression in HepG2 Liver Carcinoma Cells

“…Transiently transfected HepG2 cells were lysed in radioimmunoprecipitation assay buffer, and then approximately 60 g of total protein was resolved on NuPage 4 to 12% Bis-Tris gels (Invitrogen) and transferred to nitrocellulose membranes. The membranes were blocked for 2 h and then incubated overnight at 4°C with primary antibodies against PXR (rabbit polyclonal anti-hPXR serum (Saradhi et al, 2005) at a 1:10,000 dilution; PP2C␤l (rabbit polyclonal anti-PP2C␤; Bethyl Laboratories, Montgomery, TX) at a 1:2000 dilution; or actin (mouse monoclonal anti-␤-actin; Sigma-Aldrich) at a 1:5000 dilution. Infrared dye-conjugated secondary goat antibodies (anti-rabbit or anti-mouse) (LI-COR Biosciences, Lincoln, NE) were used at a 1:10,000 dilution, and antigen-antibody interactions were visualized using an Odyssey infrared imager (LI-COR Biosciences).…”

Pregnane X Receptor in Drug Development

“…However, as with many other NRs, PXR proves to be a difficult protein to purify. Full-length recombinant hPXR expressed in E. coli cells was first purified as inclusion bodies, followed by resolubilization, to be used for the generation of polyclonal antibodies (Saradhi et al 2005). Aside from this single report, only the LBD of PXR is commonly expressed and purified from bacterial systems to be used in biochemical assays and crystallographic experiments.…”