Faculty of Chemistry and Chemical Technology, Department of Chemistry and Biochemistry, University of Ljubljana, Ljubljana, Slovenia.
Department of Chemistry, Umeå University, Umeå, Sweden.
Methods Mol Biol. 2022;2447:1-11. doi: 10.1007/978-1-0716-2079-3_1.
Type I metacaspases are the most ubiquitous of the three metacaspase types and are present in representatives of prokaryotes, unicellular eukaryotes including yeasts, algae, and protozoa, as well as land plants. They are composed of two structural units: a catalytic so-called p20 domain with the His-Cys catalytic dyad and a regulatory p10 domain. Despite their structural homology to caspases, these proteases cleave their substrates after the positively charged amino acid residues at the P1 position, just like the metacaspases of type II and type III. We present a protocol for expression and purification of the only type I protease from a secondary endosymbiosis Guillardia theta , GtMCA-I by overexpression of its gene in BL21 (DE3) E. coli cells and one-day sequential purification using nickel-affinity, ion-exchange, and size-exclusion chromatography.
I 型 metacaspases 是三种 metacaspase 类型中最普遍的，存在于原核生物、单细胞真核生物（包括酵母、藻类和原生动物）以及陆地植物的代表中。它们由两个结构单元组成：一个催化结构域 p20，具有 His-Cys 催化二联体，以及一个调节结构域 p10。尽管它们在结构上与 caspase 同源，但这些蛋白酶在 P1 位置的正电荷氨基酸残基之后切割其底物，就像 II 型和 III 型的 metacaspases 一样。我们提出了一种通过在 BL21（DE3）E. coli 细胞中转基因表达其基因，并使用镍亲和、离子交换和分子筛层析进行为期一天的连续纯化，从二次内共生 Guillardia theta 中表达和纯化唯一的 I 型蛋白酶 GtMCA-I 的方案。
