Cyst merozoites of the protozoan parasite
Sarcocystis muris (Apicomplexa) contain three major microneme proteins at least with apparent molecular masses in the range of 15kDa. The polypeptides are lectins with affinity to galactose and some of its derivatives. At up to a concentration of 10 µg/ml of the lectin fraction no stimulation of [
3H]-thymidine incorporation was detected for spleen cell populations isolated from uninfected mice, which serve as natural intermediate host of
Sarcocystis muris. However, the lectin fraction reduced stimulation of splenocytes that were responsive to Concanavalin A. Two of the proteins mentioned above had been cloned and sequenced. The deduced molecular masses are 15.1 kDa and 15 kDa, respectively. We report here on the expression, purification, and functional analysis of the 15.1 kDa microneme protein. The polypeptide was expressed in a non-native state in
Escherichia coli inclusion bodies as a fusion protein with a poly(His) leader. The fusion protein could be purified by metal affinity chromatography and renatured in vitro. After refolding, the leader peptide was removed by proteolysis with enterokinase. Final purification of biologically active recombinant lectin molecules was achieved by lactose affinity chromatography. The identity of the recombinant protein could be confirmed by western blot analysis with a monoclonal antibody, known to be directed against one of the major microneme antigens at least. With regard to the influences on murine splenocytes, the recombinant protein seems to have the same effects like the native lectin fraction.
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