J Integr Plant Biol. ›› 2000, Vol. 42 ›› Issue (4): 383-387.

• Research Articles • Previous Articles     Next Articles

Studies on Crystalline Growth of MoFe Protein from a nifZ Deleted Strain of Azotobacter vinelandii

HUANG Ju-Fu, WANG Yao-Ping, DONG Zhi-Gang, HUANG Xiao-Ming, WANG Dao-Yong, Lv Yu-Bing, WANG Zhi-Ping   


Under a given condition of crystallization, dark brown short rhombohedron crystals could be obtained from ΔnifZ MoFe protein purified from a nifZ deleted mutant strain of Azotobacter vinelandii Lipmann. Systematic studies on the effect of concentrations of PEG 8000,MgCl2, NaCl,Tris and buffer pH on the crystallization and crystal growth of the protein showed that the protein could not be crystallized in lower concentrations of the chemicals and lower buffer pH. A large amount of smaller crystals of the protein appeared in a week with gradual increasing in the chemical concentrations and pH≥8.0. When the chemical concentrations were further increased, the time for crystallization was increased and a few high grade crystals of larger size were formed. If the concentrations of the chemicals were continuously increased, many crystals with smaller size, and, sometimes of poor quality appeared again and eventually ceased to produce any crystals. The optimal concentration for each of the above mentioned chemicals varies with other variable factors. Only one bigger crystal (both of the longest two sides: 0.16 mm) could be obtained in a hanging drop of protein sample when the concentrations of PEG 8000, MgCl2, NaCl,Tris and protein were kept at 1.86%, 300 mmol/L, 400 mmol/L, 53 mmol/L and 4.64 g/L , respectively, with Tris buffer pH 8.2.

黄巨富1 王耀萍2  董志刚1 黄孝明1  汪道涌 吕玉兵1  汪志平1

(1.  中国科学院植物研究所,北京100093;2.  中国科学院生物物理研究所,北京100101)

摘要:在一定的结晶条件下 ,缺失nifZ的棕色固氮菌 (Azotobactervinelandii Lipmann)突变种钼铁蛋白将从溶液中结晶出深棕色的短斜四棱柱晶体。PEG 80 0 0、MgCl2 、NaCl、Tris的浓度及缓冲液的pH对该蛋白的结晶及晶体生长影响的系统研究表明 ,浓度和pH较低时 ,不出晶体 ;随着浓度逐渐提高及pH高于 8.0 ,在一周内出现大量小晶体 ;再提高浓度 ,便延长结晶时间但出质好、体积大而数少的晶体 ;然后晶体随浓度的提高而又变小、变多、甚至晶质变差 ,直至不再出晶体。影响晶体生长的各因子的最适浓度随其他条件的改变而有所不同。当缓冲液的pH为 8.2而PEG 80 0 0、MgCl2 、NaCl、Tris和蛋白质的浓度分别为 1.86 %、30 0mmol/L、40 0mmol/L、5 3mmol/L和 4.6 4g/L ,在一滴悬滴液中只有一颗较大的优质晶体 (最大两边线均为 0 .16mm)。

关键词: 棕色固氮菌;缺失nifZ 的突变种;MoFe 蛋白;晶体生长

Key words: Azotobacter vinelandii, nifZ deletion mutant, MoFe protein, crystalline growth

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