Efficient detection of eukaryotic calcium-sensing receptor (CaSR) by polyclonal antibody against prokaryotic expressed t
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ORIGINAL ARTICLE
Efficient detection of eukaryotic calcium‑sensing receptor (CaSR) by polyclonal antibody against prokaryotic expressed truncated CaSR Aghdas Ramezani1 · Mohammad Javad Rasaee1 · Amirmohsen Jalaeefar2 · Ali Hatef Salmanian3 Received: 26 February 2020 / Accepted: 15 September 2020 / Published online: 1 October 2020 © Springer Nature B.V. 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. Keywords In silico analyses · CaSR · Polyclonal antibody · Immunohistofluorescence
Introduction The calcium-sensing receptor (CaSR) belongs to a subgroup of the G protein–coupled receptor (GPCR) superfamily known as the class C, family 3 or glutamate family [1]. Eight metabotropic glutamate receptors (mGluR1-8), CaSR, gamma-aminobutyric acid (GABA) receptors, a promiscuous l-α-amino acid receptor (GPRC6A), several taste and pheromone receptors [2–7] are the prominent members of this family. The glutamate family’s large amino-terminal extracellular domain (ECD) resembles a bilobed Venus flytrap (VFT) domain that is structurally similar to bacterial * Mohammad Javad Rasaee [email protected] 1
Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Jalal Ale Ahmad Highway, PO Box 14115‑331, Tehran, Iran
2
Department of Surgical Oncology, Cancer Institute, Tehran University of Medical Sciences, Tehran, Iran
3
Departments of Plant Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran
periplasmic amino acid-binding proteins [8]. For the first time, in 1993, CaSR was cloned from a cDNA library of the bovine parathyroid gland [9]. Human CaSR (hCaSR) was cloned from a woman adenomatous parathyroid gland in 1995 [10]. Other studies demonstrated that CaSR is expressed in parathyroid, kidney
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