Expression and purification of recombinant and native calreticulin.
Calreticulin is a 60-kDa Ca(2+)-binding protein of the endo(sarco)plasmic reticulum membranes of a variety of cellular systems. The protein binds approximately 25 mol of Ca2+ with low affinity and approximately 1 mol of Ca2+ with high affinity and is believed to be a site for Ca2+ binding/storage in the lumen of the endo(sarco)plasmic reticulum. In the present study, we describe purification procedures for the isolation of recombinant and native calreticulin. Recombinant calreticulin was expressed in Escherichia coli, using the glutathione S-transferase fusion protein system, and was purified to homogeneity on glutathione-Sepharose followed by Mono Q FPLC chromatography. A selective ammonium sulfate precipitation method was developed for the purification of native calreticulin. The protein was purified from ammonium sulfate precipitates by diethylaminoethyl-Sephadex and hydroxylapatite chromatography procedures, which eliminates the need to prepare membrane fractions. The purification procedures reported here for recombinant and native calreticulin yield homogeneous preparations of the proteins, as judged by the HPLC reverse-phase chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified native and recombinant calreticulin were identified by their NH2-terminal amino acid sequences, by their Ca2+ binding properties, and by their reactivity with anticalreticulin antibodies.
Baksh
S
S
Cardiovascular Disease Research Group, University of Alberta, Edmonton, Canada.
Burns
K
K
Busaan
J
J
Michalak
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D037282:Calreticulin; D004285:Dogs; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D009132:Muscles / Q000737:chemistry; D010179:Pancreas / Q000737:chemistry; D011487:Protein Conformation; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D002118:Calcium