Costa Mendonça-Natividade Flávia, Ricci-Azevedo Rafael, de Oliveira Thomaz Sandra Maria, Roque-Barreira Maria Cristina
Laboratory of Immunochemistry and Glycobiology, Department of Cell and Molecular Biology and Pathogenic Bioagents, Ribeirão Preto Medical School, University of São Paulo (FMRP/USP), Ribeirão Preto, SP, Brazil.
Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil.
Methods Mol Biol. 2020;2132:391-400. doi: 10.1007/978-1-0716-0430-4_38.
Some lectins of pathogens interact with host cells through the recognition of specific carbohydrates displayed on the mammals' cell surface. The microneme protein 1 (MIC1) from Toxoplasma gondii has a lectin domain that specifically binds sialic acid residues, often found in the terminal positions of N-glycans of mammalian cells. The necessary studies on the MIC1 biological roles have been limited initially by the laborious purification of the protein from T. gondii tachyzoites and the low yields verified. Then Escherichia coli has been transformed with a construct containing the MIC1 gene, and the obtained recombinant MIC1 (rMIC1) has been purified from the inclusion bodies. Herein, we detail the methodology of heterologous production and purification of rMIC1 and protocols to assay the rMIC1 lectin ability.
一些病原体的凝集素通过识别哺乳动物细胞表面展示的特定碳水化合物与宿主细胞相互作用。来自刚地弓形虫的微线体蛋白1（MIC1）具有一个凝集素结构域，该结构域特异性结合唾液酸残基，唾液酸残基通常位于哺乳动物细胞N-聚糖的末端位置。对MIC1生物学作用的必要研究最初受到从刚地弓形虫速殖子中费力纯化该蛋白以及验证的低产量的限制。然后用含有MIC1基因的构建体转化大肠杆菌，并从包涵体中纯化得到的重组MIC1（rMIC1）。在此，我们详细介绍了rMIC1的异源生产和纯化方法以及检测rMIC1凝集素能力的方案。
