Expression and purification of mammalian calreticulin in Pichia pastoris.

Journal article

Calreticulin is a 46-kDa Ca(2+)-binding chaperone of the endoplasmic reticulum membranes. The protein binds Ca(2+) with high capacity, affects intracellular Ca(2+) homeostasis, and functions as a lectin-like chaperone. In this study, we describe expression and purification procedures for the isolation of recombinant rabbit calreticulin. The calreticulin was expressed in Pichia pastoris and purified to homogeneity by DEAE-Sepharose and Resource Q FPLC chromatography. The protein was not retained in the endoplasmic reticulum of Pichia pastoris but instead it was secreted into the external media. The purification procedures reported here for recombinant calreticulin yield homogeneous preparations of the protein by SDS-PAGE and mass spectroscopy analysis. Purified calreticulin was identified by its NH(2)-terminal amino acid sequences, by its Ca(2+) 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 Ca(2+)-binding properties of the recombinant protein indicated that it was correctly folded.

Authors: Andrin, C; Corbett, E; Johnson, S; Dabrowska, M; Campbell, I

• Publication status: Published
• Journal: Protein expression and purification
• Volume: 20
• Issue: 2
• Pages: 207-215
• Publication date: 2000-11-01
• DOI: 10.1006/prep.2000.1291
• EISSN: 1096-0279
• ISSN: 1046-5928
• Language: English
• Keywords: Ribonucleoproteins; Animals; Sequence Analysis, Protein; Microscopy, Fluorescence; Calreticulin; Recombinant Proteins; Amino Acid Sequence; Rabbits; Calcium Radioisotopes; Pichia; Spectrometry, Mass, Electrospray Ionization; Protein Sorting Signals; Molecular Sequence Data; Disulfides; Protein Folding; Circular Dichroism; Calcium-Binding Proteins; Retina; Dogs; Electrophoresis, Polyacrylamide Gel