【摘要】 目的 :探讨野生型及点变异型牙龈卟啉单胞菌FtsZs体外聚合的特性。方法 :将质粒 pEZ1(携带野生型PgFtsZ基因的质粒 )、pYW 7(ZA32 0H ,携带野生型PgFtsZ第 32 0位的丙氨酸由组氨酸取代的点变异型PgFtsZ基因的质粒 )和 pYW 8(ZA32 0R ,携带野生型PgFtsZ第 32 0位的丙氨酸由精氨酸取代的点变异型PgFtsZ基因的质粒 )导入EscherichiacoliBL2 1(DE3)pLysS中 ,通过IPTG诱导表达目的蛋白质 ,6mol/L尿素变性及HiTrapSP层析柱纯化目的蛋白质 ,最后通过沉淀法检测FtsZ体外聚合的特性。结果 :仅 10mmol/LMgCl2 就能诱导野生型PgFtsZ、ZA32 0H和ZA32 0R的体外聚合 ,而与添加的GTP无关。结论 :野生型PgFtsZ体外聚合完全不同与E .coliFtsZ绝对依赖于GTP的特性 ,ZA32 0H和ZA32 0R体外聚合特性与野生型PgFtsZ相同 ,表明PgFtsZ的第 32 0位的丙氨酸与其体外聚合功能无关。更多还原

【Abstract】 Objective: To investigate assembly of the wild-type and the point mutant FtsZs from Porphyromonas gingivalis in vitro. Methods: The plasmids pEZ1 (containing the wild-type PgFtsZ gene)、pYW7 (ZA320H, containing a point mutant PgFtsZ gene, viz., the wild-type PgFtsZ replaced Ala320 with histidine) and pYW8 (ZA320R, containing a point mutant PgFtsZ gene, viz., the wild-type PgFtsZ replaced Ala320 with arginine) were introduced into Escherichia coli BL21(DE3)pLysS cells, and overexpressed with isopropyl-β-D-thiogalactopyranoside (IPTG). Proteins were denatured with 6 M urea, and purified by a column of HiTrap SP. Assembly of the wild-type PgFtsZ、ZA320H and ZA320R was detected by sedimentation. Result: 10 mM of MgCl 2 induced polymerization of the wild-type PgFtsZ、ZA320H and ZA320R irrespective of the nature of the added nucleotide (GTP) in vitro. Conclusions: Assembly of the wild-type PgFtsZ is different from E. coli FtsZ polymerization that absolutely requires GTP in vitro. ZA320H and ZA320R have the same characterization of their assembly in vitro, like the wild-type PgFtsZ. Therefore, Ala320 residue of PgFtsZ is not required for its assembly in vitro.更多还原