High-level bacterial expression, purification and characterization of human calreticulin.
To investigate its cellular function and role in autoimmune disease pathogenesis, we have bacterially expressed human calreticulin, a major calcium-binding protein in the endoplasmic reticulum and a human autoantigen. This is the first report describing the heterologous expression of calreticulin from any source. The recombinant calreticulin constituted approximately 32% of the soluble Escherichia coli proteins, and was purified to apparent homogeneity by ion exchange and hydrophobic liquid chromatography. As does the bona fide protein, the recombinant calreticulin binds calcium and undergoes changes in its conformation upon Zn2+ binding. We take this as a strong indication that the folding of the E.coli-expressed calreticulin is very similar, if not identical, to that of the authentic protein. Moreover, the bacterially expressed calreticulin readily reacted with anti-human and anti-rabbit antibodies, and the anti-recombinant calreticulin antibodies immunoreacted with HeLa calreticulin. The availability of this expression system will allow us to carry out site-specific and deletion mutagenesis analysis in structure--function studies of calreticulin.
Rokeach
L A
LA
Agouron Institute, La Jolla, CA 92037.
Haselby
J A
JA
Hoch
S O
SO
D000595:Amino Acid Sequence; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D037282:Calreticulin; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D015032:Zinc / Q000378:metabolism
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D011994:Recombinant Proteins; D015032:Zinc; D002118:Calcium