N末端的缺失对牙龈卟啉单胞菌FtsZ体外聚合的影响

Effects of N-terminus Deleted Mutants on Assembly of FtsZ in Porphyromonas Gingivalis in Vitro

目的:探讨N末端的缺失对牙龈卟啉单胞菌FtsZ体外聚合的影响。方法:将质粒pEZ1(PgFtsZ,携带野生型牙龈卟啉单胞菌FtsZ的基因)、pYWN1(ZΔN01,携带PgFtsZ的N末端去除了43个保守性氨基酸残基的缺失变异型PgFtsZ的基因)和pYWN2(ZΔN02,携带PgFtsZ的N末端去除了205个保守性氨基酸残基的缺失变异型Pg-FtsZ的基因)分别导入Escherichia coli BL21(DE3)pLysS中表达,通过IPTG诱导表达目的蛋白质,6 mol/L尿素变性及HiTrap SP层析柱纯化目的蛋白质,最后通过沉淀法检测10 mmol/L的MgCl2、CaCl2和MnCl2对PgFtsZ、ZΔN01和ZΔN02体外聚合的影响。结果:10 mmol/L的MgCl2、CaCl2和MnCl2均能诱导PgFtsZ和ZΔN01出现聚合反应,然而10 mmol/L的MgCl2和CaCl2没能引起ZΔN02的体外聚合,而仅有10 mmol/L的MnCl2能诱导ZΔN02出现聚合反应,但效果不如PgFtsZ和ZΔN01明显。结论:PgFtsZ的N末端的43个氨基酸残基对Mg2+、Ca2+和Mn2+诱导其体外聚合反应影响不大,而后的162个氨基酸残基在PgFtsZ的体外聚合过程中起着关键的作用。

Objective ： To investigate the effects of N - terminus deleted mutants on assembly of FtsZ in Porphyromonas gingivalis in vitro. Methods： The plasmids pEZ1 （PgFtsZ, containing FtsZ gene of Porphyromonas gingivalis）, pYWN1 （ZΔN01, containing N -terminus truncated mutant gene, missing 43 residues from the N -terminus of PgFtsZ）, pYWN2 （ ZΔN02, containing N - terminus truncated mutant gene, missing 205 residues from the N - terminus of PgFtsZ） were introduced into Escherichia coli BL21 （ DE3 ） pLysS cells, respectively, and overexpressed with isopropyl - β- D - thiogalactopyranoside （IPTG）. Proteins were denatured with 6 M urea, and purified by a column of HiTrap SP. Polymerizations of PgFtsZ, ZΔN01, ZΔN02 were detected by sedimentation in the presence of 10 mM of MgC12 , CaC12 , and MnC12 , respectively. Results： Addition of 10 mM of MgCl2, CaCl2, and MnCl2 led to the polymerization of ZΔN01, like the PgFtsZ. However, only addition of 10 mM of MnCl2 was able to induce the assembly of ZΔN02. Conclusion： 43 residues from the N - terminus of PgFtsZ have no important role in cation - induced （ Mg^2 ＋ , Ca^2 ＋ , Mn^2 ＋ ） polymerization in vitro. However, 162 residues, between 43 and 205 residues from the N -terminus are important for assembly of PgFtsZ in vitro.