Expression, purification, and biochemical characterization of a recombinant lectin of Sarcocystis muris (Apicomplexa) cyst merozoites.
The mature major microneme protein of Sarcocystis muris cyst merozoites, which is known as a dimeric lectin with high affinity to galactose and some of its derivatives, was expressed in Escherichia colias a histidine-tagged fusion protein. The recombinant polypeptide, which was recognized by a monoclonal antibody directed against the native lectin, was purified from inclusion bodies after solubilization and refolding, using a combination of metal chelate and lactose affinity chromatography. The apparent molecular mass of the refolded polypeptide as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoreses was 16 kDa, whereas gel filtration chromatography clearly demonstrated that the recombinant protein, like its native counterpart, exists as a homodimer of two non-covalently associated subunits. Inhibition of haemagglutination suggests that the combining site of the recombinant lectin recognizes N-acetyl-galactosamine as the dominant sugar, thus confirming the correct folding of the monosaccharide combining site in the renatured lectin. To the best of our knowledge, this work represents the first reported detailed characterization of a recombinant lectin from apicomplexan parasites, and may contribute to a better understanding of the process of host cell recognition and invasion by these obligate intracellular protozoa.
Klein
H
H
Paul-Ehrlich-Institut, Federal Agency for Sera and Vaccines, FG Parasitologie/Diagnostika, Langen, Germany. HARALD.KLEIN@EM.UNI-FRANKFURT.DE
Löschner
B
B
Zyto
N
N
Pörtner
M
M
Montag
T
T
D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000235:genetics; D006386:Hemagglutination Tests; D006801:Humans; D007785:Lactose; D037102:Lectins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012522:Sarcocystis / Q000737:chemistry*
D000953:Antigens, Protozoan; D037102:Lectins; D011994:Recombinant Proteins; D007785:Lactose