Purification and characterization of calreticulin: a Ca²⁺-binding chaperone from sheep kidney.

Abstract

Calreticulin (CRT) is a molecular chaperone with a molecular mass of 46 kDa present in the endoplasmic reticulum (ER). This protein is primarily involved in the regulation of intracellular Ca(2+) homeostasis and Ca(2+) storage in the ER. CRT also plays a significant role in autoimmunity and cancer. This protein contains three distinct structural domains with specialized functions. Here, we are reporting a simple procedure for the purification of CRT from mammalian kidney. To isolate CRT, sheep kidney was crushed and kept for 12 h in the extraction buffer. The lysate was centrifuged, and supernatant was precipitated by ammonium sulphate. The precipitate of 90 % ammonium sulphate was extensively dialyzed and loaded on DEAE-Hi-Trap FF and Mono Q chromatography columns. The purity of CRT was confirmed by SDS-PAGE. Finally, the protein was identified by matrix-assisted laser desorption/ionization time of flight. The purified protein was further characterized for secondary structural elements using the far-UV circular dichroism measurements. Our purification procedure is fast and simple with high yield.