Expression and purification of mammalian calreticulin in Pichia pastoris

Calreticulin is a 46-kDa Ca2+-binding chaperone of the endoplasmic reticulu m membranes. The protein binds Ca2+ with high capacity, affects intracellul ar Ca2+ homeostasis, and functions as a lectin-like chaperone. In this stud y, we describe expression and purification procedures for the isolation of recombinant rabbit calreticulin. The calreticulin was expressed in Pichia p astoris and purified to homogeneity by DEAE-Sepharose and Resource Q FPLC c hromatography. The protein was not retained in the endoplasmic reticulum of Pichia pastoris but instead it was secreted into the external media. The p urification procedures reported here for recombinant calreticulin yield hom ogeneous preparations of the protein by SDS-PAGE and mass spectroscopy anal ysis. Purified calreticulin was identified by its NH2-terminal amino acid s equences, by its Ca2+ binding, and by its reactivity with anti-calreticulin antibodies. The protein contained one disulfide bond between (88)Cys and ( 120)Cys. CD spectral analysis and Ca2+-binding properties of the recombinan t protein indicated that it was correctly folded. (C) 2000 Academic Press.