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Protein Engineering vol. 4 no. 8 pp. 981-987, 1991
© 1991 Oxford University Press

RESEARCH-ARTICLE

High-level bacterial expression, purification and characterization of human calreticulin

Luis A. Rokeach1,2, Jeanne A. Haselby and Sallie O. Hoch

The Agouron Institute 505 Coast Blvd South, La Jolla, CA 92037, USA 2Present address: Département de Biochimie, Faculté de Médecine, Université de Montréal Montréal, Québec, Canada H3C 3J7

1To whom correspondence should be addressed

To investigate its cellular function and role in autoimmune disease pathogenesis, we have bacterlally 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 {small tilde}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.

Keywords: autoantigen/calcium binding/onchocerciasis/sitespecific mutagenesis/two-cistron expression

Received August 14, 1991; accepted September 23, 1991.


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