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Updated: Aug 15, 2026
Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System
Published on: May 1, 2019
Purification of full-length human pregnane and xenobiotic receptor: polyclonal antibody preparation for immunological
Mallampati Saradhi1, Biji Krishna, Gauranga Mukhopadhyay
1Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi 110067, India.
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.
Insights
Researchers successfully produced and purified the Pregnane and Xenobiotic Receptor (PXR) in bacteria. An antibody against PXR was developed, proving effective for detecting PXR in human and mouse cells and tissues.
Area of Science:
- Molecular biology
- Biochemistry
- Immunology
Background:
- Pregnane and Xenobiotic Receptor (PXR) is a nuclear receptor involved in xenobiotic metabolism and elimination.
- Understanding PXR's role in homeostasis requires reliable tools for its detection and study.
Purpose of the Study:
- To achieve prokaryotic expression and purification of full-length PXR.
- To generate a polyclonal antibody against PXR for its detection and characterization.
- To investigate PXR's localization and expression in cells and tissues.
Main Methods:
- Full-length PXR cDNA was cloned into a prokaryotic expression vector and overexpressed in E. coli.
- Recombinant PXR was purified using affinity chromatography.
- An antibody against PXR was generated and validated through immunocytology, Western blot, EMSA, and immunohistochemistry.
Main Results:
- Successful expression and purification of full-length PXR.
- Development of a specific and sensitive polyclonal antibody against PXR.
- Demonstrated nuclear localization of PXR in transfected cells and endogenous PXR in liver tissues.
- Identified a 50 kDa band for endogenous PXR in HepG2 and COLO320DM cells.
Conclusions:
- The generated anti-PXR antibody is a valuable tool for detecting human and mouse PXR.
- The antibody facilitates the study of PXR localization and expression, aiding functional characterization.
- This research paves the way for potential clinical applications of PXR research.
