Crystal structure of a binary complex between human GCN5 histone acetyltransferase domain and acetyl coenzyme A
Crystal structure of a binary complex between human GCN5 histone acetyltransferase domain and acetyl coenzyme A
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DOI：
10.1002/prot.21407
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发表时间：
2007-07-01
影响因子：
2.9
通讯作者：
Plotnikov, Alexander N.
中科院分区：
文献类型：
--
作者：
Schuetz, Anja;Bernstein, Galina;Plotnikov, Alexander N.
材料和方法。蛋白质表达和纯化：通过 PCR 从哺乳动物基因保藏克隆（登录号 gi：10835101）中扩增编码人 GCN5 氨基酸 497-662 的 DNA，并将其亚克隆到修饰的 pET28a-LIC 载体中（详细信息请参见 http://www.sgc.utoronto.ca/SGC-WebPages/toronto-vectors.php）。重组蛋白在大肠杆菌 BL21 (DE3) Codon plus RIL (Stratagene) 中过表达为 N 端 His6 标签蛋白。细胞在50 lg/mL卡那霉素存在下在Terrific Broth中于378℃生长至约0.8的OD600，用0.5mM异丙基-1-硫代-D-吡喃半乳糖苷诱导，并于158℃温育过夜。为了纯化，将细胞糊重悬于裂解缓冲液（50 mM 磷酸钠/磷酸钾缓冲液，pH 7.5，500 mM NaCl，5% 甘油和 0.1 mM 苯甲基磺酰氟）中。通过在 18,000 psi 下通过 Microfluidizer (Microfluidics Corporation) 裂解细胞。通过离心使裂解物澄清，并将上清液加到装有 Ni2+ 的 5 mL HiTrap 螯合柱（Amersham Biosciences）上。用 50 mL 20 mM HEPES-NaOH、pH 7.5 缓冲液（含有 500 mM NaCl 和 50 mM 咪唑）洗涤柱。用 20 mM HEPES-NaOH、pH 7.5、500 mM NaCl、5% 甘油和 250 mM 咪唑洗脱蛋白质。为了切割His标签，将凝血酶(Sigma)添加到含有GCN5的级分中，并将样品在48°C下孵育过夜，同时针对20 mM HEPES-NaOH、pH 7.5和150 mM NaCl进行透析。使用阳离子交换层析在Source 30 S柱(10 3 10)(Amersham Biosciences)上进一步纯化蛋白质至同质，用20 mM HEPES-NaOH，pH 7.5平衡。 GCN5 使用高达 500 mM NaCl 的线性梯度（30 倍柱体积）进行洗脱。纯化产量为每 1 L 培养物 20 mg 蛋白质。 蛋白质结晶：纯化的人 GCN5 HAT 结构域与 AcCoA 以蛋白质与辅酶 1:5 的摩尔比复合，并使用坐滴蒸汽扩散法在 208°C 下结晶，将 1 µL 8.4 mg/mL 蛋白质溶液与 1 µL 含有 15%(v/v) 乙醇和 100 mM 的储库溶液混合Tris-HCl，pH 7.0。在液氮中快速冷冻晶体之前，
Materials and Methods. Protein expression and purification: DNA encoding amino acids 497–662 of human GCN5 was amplified by PCR from the Mammalian Gene Collection clone (accession code gi: 10835101) and subcloned into a modified pET28a-LIC vector (for details see http://www. sgc. utoronto. ca/SGC-WebPages/toronto-vectors. php). The recombinant protein was overexpressed as an N-terminal His6-tagged protein in E. coli BL21 (DE3) Codon plus RIL (Stratagene). Cells were grown in Terrific Broth in presence of 50 lg/mL kanamycin at 378C to an OD600 of about 0.8, induced with 0.5 mM isopropyl-1-thio-D-galactopyranoside, and incubated overnight at 158C. For purification cell paste was resuspended in lysis buffer (50 mM sodium/potassium phosphate buffer, pH 7.5, 500 mM NaCl, 5% glycerol, and 0.1 mM phenylmethyl sulfonyl fluoride). Cells were lysed by passing through Microfluidizer (Microfluidics Corporation) at 18,000 psi. The lysate was clarified by centrifugation and the supernatant was applied to a 5 mL HiTrap Chelating column (Amersham Biosciences), charged with Ni2þ. The column was washed with 50 mL of 20 mM HEPES-NaOH, pH 7.5 buffer, containing 500 mM NaCl and 50 mM imidazole. The protein was eluted with 20 mM HEPES-NaOH, pH 7.5, 500 mM NaCl, 5% glycerol, and 250 mM imidazole. To cleave the His-tag, thrombin (Sigma) was added to GCN5 containing fractions and the sample was incubated overnight at 48C while dialyzing against 20 mM HEPES-NaOH, pH 7.5, and 150 mM NaCl. The protein was further purified to homogeneity using cation exchange chromatography on Source 30 S column (10 3 10)(Amersham Biosciences), equilibrated with 20 mM HEPES-NaOH, pH 7.5. GCN5 was eluted with a linear gradient of up to 500 mM NaCl (in 30 column volumes). Purification yield was 20 mg of protein per 1 L of culture.Protein crystallization: Purified HAT domain of human GCN5 was complexed with AcCoA at 1: 5 molar ratio of protein to coenzyme, and crystallized using the sitting drop vapor diffusion method at 208C by mixing 1 lL of 8.4 mg/mL protein solution with 1 lL of the reservoir solution containing 15%(v/v) ethanol and 100 mM Tris-HCl, pH 7.0. Before flash-freezing the crystal in liquid nitrogen,