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Efficient Detection of Eukaryotic Calcium-Sensing Receptor (Casr) by Polyclonal Antibody Against Prokaryotic Expressed Truncated Casr Publisher Pubmed



Ramezani A1 ; Rasaee MJ1 ; Jalaeefar A2 ; Salmanian AH3
Authors
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Authors Affiliations
  1. 1. Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Jalal Ale Ahmad Highway, PO Box 14115-331, Tehran, Iran
  2. 2. Department of Surgical Oncology, Cancer Institute, Tehran University of Medical Sciences, Tehran, Iran
  3. 3. Departments of Plant Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran

Source: Molecular Biology Reports Published:2020


Abstract

Calcium-sensing receptor (CaSR), which is better known for its action as regulating calcium homeostasis, can bind various ligands. To facilitate research on CaSR and understand the receptor's function further, an in silico designed truncated protein was developed. The resulting protein folding indicated that 99% of predicted three dimensional (3D) structure residues are located in favored and allowed Ramachandran plots. However, it was found that such protein does not fold properly when expressed in prokaryotic host cells. Thioredoxin (Trx) tag was conjugated to increase the final protein's solubility, which could help obtain the soluble antigen with better immunogenic properties. The truncated recombinant proteins were expressed and purified in two forms (Trx-CaSR: RR19 and CaSR: RRJ19). The polyclonal antibody was induced by the rabbit immunization with the form of RR19. Western blot on mouse kidney lysates evidenced the proper immune recognition of the receptor by the produced antibody. The specificity and sensitivity of antibodies were also assayed by immunohistofluorescence. These experiments affirmed antibody's ability to indicate the receptor on the cell surface in native form and the possibility of applying such antibodies in further cellular and tissue assays. © 2020, Springer Nature B.V.
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