Wilharm Gottfried, Neumayer Wibke, Heesemann Jürgen
Department of Bacteriology, Max von Pettenkofer-Institut, Pettenkoferstr 9a, Munich D-80336, Germany.
Protein Expr Purif. 2003 Oct;31(2):167-72. doi: 10.1016/s1046-5928(03)00183-9.
Pathogenic Yersinia species (Y. enterocolitica, Y. pestis, and Y. pseudotuberculosis) make use of a virulence plasmid-encoded type three secretion system (TTSS) to inject effector proteins into host cells. Y. enterocolitica YscM1 (LcrQ in Y. pestis and Y. pseudotuberculosis) and its homologue YscM2 are regulatory components of the TTSS that are also secreted by this transport apparatus. YscM1 and YscM2 share 57% identity and are believed to be functionally equivalent. We have recombinantly expressed and purified YscM1 and YscM2 in Escherichia coli. After expression as glutathione S-transferase (GST) fusions purification to near homogeneity was achieved by glutathione-Sepharose affinity chromatography followed by PreScission protease treatment to cleave off GST and gel filtration on a Superdex 75 column. Such recombinant YscM1 and YscM2 bound efficiently to the specific chaperone SycH, indicating proper folding of the purified proteins. Gel filtration analyses revealed that both YscM1 and YscM2 formed homodimers. The YscM1 and YscM2 homodimers could be dissociated at high ionic strength, indicating that salt bridges essentially contribute to the dimerization. We further demonstrated that YscM1 and YscM2 are susceptible to thrombin cleavage.
致病性耶尔森菌属（小肠结肠炎耶尔森菌、鼠疫耶尔森菌和假结核耶尔森菌）利用一种毒力质粒编码的三型分泌系统（TTSS）将效应蛋白注入宿主细胞。小肠结肠炎耶尔森菌的YscM1（鼠疫耶尔森菌和假结核耶尔森菌中的LcrQ）及其同源物YscM2是TTSS的调节成分，它们也通过该转运装置分泌。YscM1和YscM2的同一性为57%，被认为在功能上是等效的。我们已经在大肠杆菌中重组表达并纯化了YscM1和YscM2。以谷胱甘肽S-转移酶（GST）融合蛋白形式表达后，通过谷胱甘肽-琼脂糖亲和层析实现了近乎均一的纯化，随后用PreScission蛋白酶处理以切割掉GST，并在Superdex 75柱上进行凝胶过滤。这种重组的YscM1和YscM2能有效地与特定伴侣蛋白SycH结合，表明纯化后的蛋白折叠正确。凝胶过滤分析表明，YscM1和YscM2均形成同源二聚体。YscM1和YscM2同源二聚体在高离子强度下可解离，表明盐桥对二聚化起重要作用。我们进一步证明YscM1和YscM2易受凝血酶切割。
