缺失nifH的棕色固氮菌突变种固氮酶钼铁蛋白的结晶

从限氨固氮培养基中培养的缺失nifH的棕色固氮菌(AzotobactervinelandiiLipmann)突变种DJ54中,分离纯化出部分纯的缺失FeMoco的钼铁蛋白(ΔnifHAv1)。用相同纯化方法分别从DJ35和UW45突变种中纯化的ΔnifEAv1和NifB-Av1的纯度明显高于ΔnifHAv1的纯度。在合适的结晶条件下,可得到这三种蛋白的深棕色短斜四棱柱晶体。ΔnifHAv1与NifB-Av1一样,晶体形成所需的时间比ΔnifEAv1的长。而它结晶所需的沉淀剂和缓冲液最适浓度则与ΔnifEAv1的相同。SDS-PAGE鉴定表明,结晶的ΔnifHAv1与OPAv1的组成相似。这表明,在ΔnifHAv1溶液中形成的晶体可能就是该蛋白质的晶体。

Crystallization of MoFe Protein (DnifH Av1) from a nifH Deleted Strain of Azotobacter vinelandii

A FeMoco-deficient DnifH Av1 was partially purified from a nifH deleted mutant DJ54 ofAzotobacter vinelandii Lipmann grown in NH3-limited medium. By using the same purification method, DnifEAv1 and NifB-Av1 were obtained from DJ35 and UW45, respectively. The latter two proteins were obviouslymuch purer than DnifH Av1. Under a suitable condition for crystallization, dark brown short rhombohedroncrystals could be obtained from the three proteins. Like NifB-Av1, the time for formation of DnifH Av1crystal was longer than that of DnifE Av1. But the optimal concentrations of precipitant and buffer forcrystallization of DnifH Av1 were similar to those of DnifE Av1. SDS-PAGE analysis showed that thecrystalline DnifH Av1 was similar in the composition to OP Av1. It indicates that the crystal formed in DnifHAv1 solution could be the protein crystal.