Expression and purification of mammalian calreticulin in Pichia pastoris.
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.
Andrin
C
C
CIHR Group in Molecular Biology of Membranes, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada.
Corbett
E F
EF
Johnson
S
S
Dabrowska
M
M
Campbell
I D
ID
Eggleton
P
P
Opas
M
M
Michalak
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002132:Calcium Radioisotopes; D002135:Calcium-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D037282:Calreticulin; D002942:Circular Dichroism; D004220:Disulfides / Q000378:metabolism; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D017510:Protein Folding; D021382:Protein Sorting Signals; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012160:Retina / Q000166:cytology; D012261:Ribonucleoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D020539:Sequence Analysis, Protein; D021241:Spectrometry, Mass, Electrospray Ionization
D002132:Calcium Radioisotopes; D002135:Calcium-Binding Proteins; D037282:Calreticulin; D004220:Disulfides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D012261:Ribonucleoproteins