Expression of calreticulin in Escherichia coli and identification of its Ca2+ binding domains.
Recombinant calreticulin and discrete domains of calreticulin were expressed in Escherichia coli, using the glutathione S-transferase fusion protein system, and their Ca2+ binding properties were determined. Native calreticulin bound 1 mol of Ca2+/mol of protein with high affinity, and also bound approximately 20 mol of Ca2+/mol of protein with low affinity. Both Ca2+ binding sites were present in the recombinant calreticulin indicating that proper folding of the protein was achieved using this system. Calreticulin is structurally divided into three distinct domains: the N-domain encompassing the first 200 residues; the P-domain which is enriched in proline residues (residue 187-317); and the C-domain which covers the carboxyl-terminal quarter of the protein (residues 310-401), and contains a high concentration of acidic residues. These domains were expressed in E. coli, isolated, and purified, and their Ca2+ binding properties were analyzed. The C-domain bound approximately 18 mol of Ca2+/mol of protein with a dissociation constant of approximately 2 mM. The P-domain bound approximately 0.6-1 mol of Ca2+/mol of protein with a dissociation constant of approximately 10 microM. The P-domain and the C-domain, when expressed together as the P+C-domain, bound Ca2+ with both high affinity and low affinity, reminiscent of both full length recombinant calreticulin and native calreticulin. In contrast the N-domain, did not bind any detectable amount of 45Ca2+. We conclude that calreticulin has two quite distinct types of Ca2+ binding sites, and that these sites are in different structural regions of the molecule. The P-domain binds Ca2+ with high affinity and low capacity, whereas the C-domain binds Ca2+ with low affinity and high capacity.
Baksh
S
S
Cardiovascular Disease Research Group, University of Alberta, Edmonton, Canada.
Michalak
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D037282:Calreticulin; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D002118:Calcium