Crystal structure of Hsp33 chaperone (TM1394) from Thermotoga maritima at 2.20 Å resolution
Crystal structure of Hsp33 chaperone (TM1394) from Thermotoga maritima at 2.20 Å resolution
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DOI：
10.1002/prot.20542
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发表时间：
2005-11-15
影响因子：
2.9
通讯作者：
Wilson, IA
中科院分区：
文献类型：
--
作者：
Jaroszewski, L;Schwarzenbacher, R;Wilson, IA
材料和方法.蛋白质生产和结晶.通过聚合酶链反应（PCR），使用PfuTurbo（Stratagene）和编码预测的5′-和3′-末端的引物对，从基因组DNA扩增来自海栖热袍菌（Thermotoga maritima）（TIGR：TM 1394，Swiss-Prot：Q9 X1 B4）的Hsp 33。将PCR产物克隆到质粒pMH 4中，其在全长蛋白质的氨基末端编码表达和纯化标签（MGSDKIHHHHH）。通过测序确认克隆连接。使用大肠杆菌菌株GeneHogs®在改良的Terrific肉汤中进行蛋白质表达。在发酵结束时向培养物中加入溶菌酶至250 μg/mL的终浓度。在裂解缓冲液[50 mM Tris pH 7.9，50 mM NaCl，10 mM咪唑，0.25 mM Tris]中进行冻融程序后，通过超声裂解细菌（2-羧乙基）膦盐酸盐（TCEP）]，将细胞碎片以3400× g离心60 min沉淀。将可溶性级分应用于镍螯合树脂（阿默舍姆Biosciences）上进行。用洗涤缓冲液[50 mM磷酸钾pH 7.8，300 mM NaCl，40 mM咪唑，10%（v/v）甘油，0.25 mM TCEP]洗涤树脂，用洗脱缓冲液[20 mM Tris pH 7.9，300 mM咪唑，10%（v/v）甘油，0.25 mM TCEP]洗脱靶蛋白。将缓冲液交换到含有50 mM NaCl的缓冲液Q [20 mM Tris pH 7.9，5%（v/v）甘油，0.25 mM TCEP]中，并施加到用相同缓冲液预平衡的RESOURCE Q柱（阿默舍姆Biosciences）上。使用缓冲液Q中50至500 mM NaCl的线性梯度洗脱靶蛋白。合并适当的RESOURCE Q级分，缓冲液交换至结晶缓冲液[20 mM Tris pH 7.9，150 mM NaCl，0.25 mM TCEP]中，并通过离心超滤（Millipore）浓缩至15 mg/mL用于结晶测定。使用1.0× 30 cm Superdex 200柱（阿默舍姆Biosciences）结合静态光散射（Wyatt Technology）测定靶蛋白的分子量和寡聚状态。移动的相由20 mM Tris pH 7.9和150 mM NaCl 0.02%（w/v）叠氮化钠组成。使用具有标准JCSG结晶方案的纳米液滴气相扩散方法5使蛋白质结晶。4.结晶试剂含有15%甘油、8.5%异丙醇、17%聚乙二醇（PEG）-4000，0.1MHEPES，pH 7.5。包括10%（v/v）乙二醇（最终浓度）作为冷冻保护剂。该晶体在斜方晶系空间群P2 1 2 1 2 1中索引（表I）。
Materials and Methods.Protein production and crystallization.Hsp33 from Thermotoga maritima (TIGR: TM1394, Swiss-Prot: Q9X1B4) was amplified by polymerase chain reaction (PCR) from genomic DNA using PfuTurbo (Stratagene) and primer pairs encoding the predicted 5′-and 3′-ends. The PCR product was cloned into plasmid pMH4, which encodes an expression and purification tag (MGSDKIHHHHHH) at the amino terminus of the full-length protein. The cloning junctions were confirmed by sequencing. Protein expression was performed in a modified Terrific Broth using the Escherichia coli strain GeneHogs®. Lysozyme was added to the culture at the end of fermentation to a final concentration of 250 μg/mL. Bacteria were lysed by sonication after a freeze/thaw procedure in Lysis Buffer [50 mM Tris pH 7.9, 50 mM NaCl, 10 mM imidazole, 0.25 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP)], and the cell debris was pelleted by centrifugation at 3400× g for 60 min. The soluble fraction was applied to a nickel-chelating resin (Amersham Biosciences) pre-equilibrated with Lysis Buffer. The resin was washed with Wash Buffer [50 mM potassium phosphate pH 7.8, 300 mM NaCl, 40 mM imidazole, 10%(v/v) glycerol, 0.25 mM TCEP], and the target protein was eluted with Elution Buffer [20 mM Tris pH 7.9, 300 mM imidazole, 10%(v/v) glycerol, 0.25 mM TCEP]. The eluate was buffer-exchanged into Buffer Q [20 mM Tris pH 7.9, 5%(v/v) glycerol, 0.25 mM TCEP] containing 50 mM NaCl and applied to a RESOURCE Q column (Amersham Biosciences) pre-equilibrated with the same buffer. The target protein was eluted using a linear gradient of 50 to 500 mM NaCl in Buffer Q. The appropriate RESOURCE Q fractions were pooled, buffer-exchanged into Crystallization Buffer [20 mM Tris pH 7.9, 150 mM NaCl, 0.25 mM TCEP], and concentrated for crystallization assays to 15 mg/mL by centrifugal ultrafiltration (Millipore). Molecular weight and oligomeric state of the target protein were determined using a 1.0× 30 cm Superdex 200 column (Amersham Biosciences) in combination with static light scattering (Wyatt Technology). The mobile phase consisted of 20 mM Tris pH 7.9 and 150 mM NaCl 0.02%(w/v) sodium azide. The protein was crystallized using the nanodroplet vapor diffusion method 5 with standard JCSG crystallization protocols. 4 The crystallization reagent contained 15% glycerol, 8.5% isopropanol, 17% polyethylene glycol (PEG)-4000, 0.1 M HEPES, pH 7.5. Ten percent (v/v) ethylene glycol (final concentration) was included as a cryoprotectant. The crystals were indexed in the orthorhombic space group P2 1 2 1 2 1 (Table I).