Expression, purification, and biochemical characterization of a recombinant Iectin of Sarcocystis muris (Apicomplexa) cyst merozoites
Expression, purification, and biochemical characterization of a recombinant Iectin of Sarcocystis muris (Apicomplexa) cyst merozoites
复制标题
鼠肉孢子虫 (Apicomplexa) 包囊裂殖子重组凝集素的表达、纯化和生化表征
作者：
H. Klein;Bettina Loschner;N. Zyto;Monika Portner;T. Montag
鼠肉孢子虫包囊裂殖子成熟的主要微线体蛋白是一种对半乳糖及其衍生物具有高亲和力的二聚体凝集素，在大肠杆菌中表达为组氨酸标记的融合蛋白。重组多肽，这是由针对天然凝集素的单克隆抗体识别，纯化包涵体溶解和重折叠后，使用金属螯合物和乳糖亲和层析的组合。由十二烷基硫酸钠-聚丙烯酰胺凝胶柱层析确定的重折叠多肽的表观分子量为16 kDa，而凝胶过滤色谱清楚地表明，重组蛋白，像它的天然对应物，作为两个非共价相关的亚基的同源二聚体存在。血凝抑制表明，重组凝集素的结合位点识别N-乙酰基-半乳糖胺作为优势糖，从而确认复性凝集素中单糖结合位点的正确折叠。据我们所知，这项工作代表了首次报道的顶复门寄生虫重组凝集素的详细表征，并且可能有助于更好地了解这些专性细胞内原生动物识别和入侵宿主细胞的过程。
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 coli as 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.