Structure of YciI from Haemophilus influenzae (HI0828) reveals a ferredoxin-like alpha/beta-fold with a histidine/aspartate centered catalytic site.
Structure of YciI from Haemophilus influenzae (HI0828) reveals a ferredoxin-like alpha/beta-fold with a histidine/aspartate centered catalytic site.
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流感嗜血杆菌 (HI0828) 的 YciI 结构揭示了铁氧还蛋白样 α/β 折叠，具有以组氨酸/天冬氨酸为中心的催化位点。
DOI：
10.1002/prot.20411
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发表时间：
2005
期刊：
影响因子：
2.9
通讯作者：
Herzberg,Osnat
中科院分区：
文献类型：
--
作者：
Willis,MarkA;Song,Feng;Zhuang,Zhihao;Krajewski,Wojciech;Chalamasetty,VaniRao;Reddy,Prasad;Howard,Andrew;Dunaway-Mariano,Debra;Herzberg,Osnat
方法.蛋白质生产。从H.流感病毒KW 20基因组DNA，并克隆到pET 15 b（Novagen）中，其含有凝血酶可切割的N-末端His 6-标签。当LB生长培养基（含100 μ g/mL氨苄青霉素）中的细胞密度达到A600 0.6时，在37 ℃下用IPTG（0.4mM终浓度）培养的大肠杆菌B834（DE 3）细胞中诱导硒代蛋氨酸取代的蛋白质表达。使用Ni-NTA柱（Qiagen）纯化蛋白质。用凝血酶切割N-末端His 6-标签，并如针对蛋白质HI 0442所述在第二Ni-NTA柱上将切割的蛋白质与未切割的蛋白质分离。1最后，将缓冲液更换为20 mM Na HEPES和2 mM咪唑（pH 7.0）、5 mM NaCl、0.1 mM EDTA和0.1 mM DTT。通过MALDI-TOF质谱法测定蛋白质的分子量和硒代蛋氨酸掺入的程度。属于空间群P212121的HI 0828的晶体（细胞尺寸为a 42.5 μ m，B 63.3 μ m，c 75.5 μ m，在不对称单位中为单个二聚体）在室温下使用等体积蛋白质的悬滴蒸气扩散实验中在几天内出现（11 mg/mL）和孔溶液（16%PEG 4000，0.1 M二甲胂酸钠，pH 5.5，20 mM
Methods. Protein production. The gene encoding HI0828 was amplified from H. influenzae KW20 genomic DNA and cloned into pET15b (Novagen), which contains a thrombin-cleavable N-terminal His6-tag. Selenomethionine-substituted protein expression was induced in Eshcerihcia coli B834 (DE3) cells cultured at 37 C with IPTG (0.4 mM final concentration) when the cell density in LB growth media (with 100 g/mL ampicillin) reached A600 0.6. The protein was purified using a Ni-NTA column (Qiagen). The N-terminal His6-tag was cleaved with thrombin, and the cleaved protein was separated from uncleaved protein on a second Ni-NTA column as described for the protein HI0442. 1 Finally, the buffer was exchanged for 20 mM Na HEPES and 2 mM imidazole (pH 7.0), 5 mM NaCl, 0.1 mM EDTA, and 0.1 mM DTT. The molecular weight of the protein and the extent of selenomethionine incorporation were determined by MALDI-TOF mass spectroscopy.Structure determination. Crystals of HI0828 belonging to space group P212121 (with cell dimensions of a 42.5 Å, b 63.3 Å, c 75.5 Å, and a single dimer in the asymmetric unit) appeared in a few days at room temperature in hanging-drop vapor diffusion experiments using equal volumes of protein (11 mg/mL), and well solution (16% PEG 4000, 0.1 M Na cacodylate, pH 5.5, 20 mM