Efficient detection of eukaryotic calcium-sensing receptor (CaSR) by polyclonal antibody against prokaryotic expressed truncated CaSR.
Mol Biol Rep
; 47(10): 7723-7734, 2020 Oct.
Article
en En
| MEDLINE
| ID: mdl-33001312
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Expresión Génica
/
Receptores Sensibles al Calcio
/
Escherichia coli
/
Anticuerpos
Tipo de estudio:
Diagnostic_studies
Límite:
Animals
Idioma:
En
Revista:
Mol Biol Rep
Año:
2020
Tipo del documento:
Article
País de afiliación:
Irán