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.
Methods Mol Biol. 2020;2132:379-389. doi: 10.1007/978-1-0716-0430-4_37.
Tachyzoites, which are infective forms of Toxoplasma gondii, use their actinomyosin system to move over surfaces and invade host cells. Central to this process is the regulated release of micronemes organelles contents. The microneme protein 4 (MIC4) has the property to recognize galactosides residues linked to glycoproteins on the host cell surface. This property allows that MIC4 binds to TLR2- and TLR4 N-linked glycans and promote the activation of cell innate immune cells and secretion of inflammatory cytokines, acting on resistance against the parasite. Obtention of MIC4 from T. gondii requires several purification steps, is time-consuming and provides low yield. Therefore, this section details the protocol for prokaryotic expression, production, and purification of recombinant MIC4 (rMIC4) and for experimental assays to confirm its biological activity.
速殖子是刚地弓形虫的感染形式，利用其肌动球蛋白系统在表面上移动并侵入宿主细胞。这个过程的核心是微线体细胞器内容物的调节释放。微线体蛋白 4（MIC4）具有识别与宿主细胞表面糖蛋白连接的半乳糖残基的特性。这种特性使得 MIC4 与 TLR2 和 TLR4 N 连接的糖结合，并促进细胞先天免疫细胞的激活和炎症细胞因子的分泌，从而抵抗寄生虫。从刚地弓形虫中获得 MIC4 需要经过几个纯化步骤，既耗时又产量低。因此，本节详细介绍了重组 MIC4（rMIC4）的原核表达、生产和纯化的方案，以及用于确认其生物学活性的实验方案。
