J Integr Plant Biol. ›› 2003, Vol. 45 ›› Issue (7): 820-824.

• Research Articles • Previous Articles     Next Articles

Crystallization of Nitrogenase MoFe Protein (NifB-Av1) from a nifB Mutated Strain UW45 of Azotobacter vinelandii

ZHAO Ying, L Yu-Bing, ZHAO Jian-Feng, ZHOU Jun-Xian, QIAN Zhong-Xing , WANG Yao-Ping, HUANG Ju-Fu   

Abstract:

Six hundred and 28 mg of NifB- Av1 was obtained by a chromatography twice on DE 52 columns and Sephacryl S-300 column from the crude extract (37 677 mg) of a nifB mutated strain UW45 of Azotobacter vinelandii Lipmann. The protein was almost homogeneous as determined by Coomassie staining of SDS gels. The analysis by SDS-PAGE showed that NifB-Av1 was similar to Av1 from wild-type strain of A. vinelandii (OP) in the kinds of subunits (αand βsubunit). When complemented with Av2, NifB-Av1 had hardly any H-reducing activity, but could be significantly activated by FeMoco extracted from Av1. Under a suitable condition for crystallization, short dark-brown rhombohedral crystals could be obtained from NifB-Av1. Both of the longest sides of the biggest crystal were 0.1 mm. The time of the formation of crystals and number, size, quality and shape of crystals obviously depended not only on the kinds and concentrations of the components in the precipitant solution, but also on the methods for crystallization and technical bias, etc. The preliminary results showed that the crystal seemed to be formed from NifB-Av1.

棕色固氮菌突变种UW45 的固氮酶钼铁蛋白(NifB-Av1)结晶
赵 颖  吕玉兵 赵剑峰  周军贤  潜忠兴  王耀萍  黄巨富
(1. 中国科学院植物研究所光合作用与环境分子生理学重点实验室,北京 100093;
2.中国科学院生物物理研究所,北京 100101)

摘要: 经两次DE52和Sephacryl S-300柱层析,从棕色固氮菌(Azotobacter vinelandii Lipmann)突变种UW45粗提液(37 677 mg蛋白)中纯化得到628 mg的NifB-Av1。经考马斯亮蓝R-250染色的SDS凝胶电泳分析表明,该蛋白基本达到SDS凝胶电泳纯,组成它的亚单位的种类与Av1(α和β亚单位)相似。NifB-Av1不能与NifB-Av2重组成具放氢活性的固氮酶,但可使与其保温重组的FeMoco显出高活性。在合适条件下,NifB-Av1可在结晶溶液中析出棕色短斜四棱柱晶体,目前所得最大晶体的二维边长均为0.1 mm。能否出现晶体以及出晶时间、晶体数目、大小、质量和形状等,与沉淀剂溶液各组分的种类和浓度、结晶方法、实验操作等因素密切相关。初步结果表明,所得晶体为NifB-Av1单晶。
关键词: 棕色固氮菌突变种UW45;固氮酶NifB-Av1;结晶

 

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