Thermodynamic and kinetic characterization of the binding of the TATA binding protein to the adenovirus E4 promoter.
Thermodynamic and kinetic characterization of the binding of the TATA binding protein to the adenovirus E4 promoter.
复制标题
TATA 结合蛋白与腺病毒 E4 启动子结合的热力学和动力学表征。
DOI：
10.1021/bi00031a020
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Brenowitz,M
中科院分区：
文献类型：
--
作者：
Petri,V;Hsieh,M;Brenowitz,M
材料和方法STBP表达和纯化。质粒pKA 9携带S.酿酒酵母SPT 15基因在pET表达载体中的表达是K. Arndt和F. Winston（Arndt等人，1992年）。开发了其纯化方案的修改，并在下文中给出。将含有质粒pLysS和pKA 9的BL 21（DE 3）细胞在37 ℃下在含有25 μ g/mL氨苄青霉素和30 μ g/mL氯霉素的LB肉汤中生长.在密度为λ = 0.4时，将肉汤转移至30 ℃，通过加入IPTG至1 mM浓度开始TBP生产，并在30 ℃下继续生长2小时。收获细胞并将细胞沉淀物储存在-70 ℃下。方案中的所有后续步骤均在4 ℃下进行。将冷冻细胞的等分试样在冰上解冻并重悬（3 mL/g细胞）在25 mM Hepes-KOH、20%甘油、1 mM EDTA、1 mM DTT（pH 7.9）中（缓冲液A）与含有抑肽酶（1 μ g/mL）、氯甲基酮在细胞重悬之前，将100 mM PMSF（20 μ g/mL）、亮抑酶肽（1 μ g/mL）、胰蛋白酶抑制剂（100 μ g/mL）和100 mM PMSF（1 mM）蛋白酶抑制剂加入缓冲液中。在Biospec Bead-Beater中使用酸洗干玻璃珠（< 106 pm，Sigma）裂解细胞，外套中填充盐饱和冰水，每隔3分钟爆发4次，每次30秒。将悬浮液在Sorvall SS-34转子中以18 000 rpm离心20 min。通过在硫酸鱼精蛋白溶液（6 mg/mL）中缓慢搅拌，使染色体DNA从上清液中沉淀，最终浓度为0.3 mg/mL。如上所述通过离心除去沉淀物。将填充有50 mL Pharmacia S-Sepharose Fast Flow树脂的2.2 x 25 cm柱在含有100 mM KCl的缓冲液A中平衡。上样后，用平衡缓冲液以4 mL/min的速度洗涤色谱柱，直至A280恢复至基线。用80 mL梯度的缓冲液A（100 - 235 mM KCl）洗脱TBP，然后用240 mL梯度的235-370 mM KCl洗脱; TBP在235 - 300 mM KCl之间洗脱。通过SDS-PAGE分析柱级分;合并含TBP的级分，使用Amicon Centricon 3浓缩器浓缩至约1.5 mL，并对含有100 mM KCl的缓冲液A透析过夜。
MATERIALS AND METHODSTBP Expression and Purification. Plasmid pKA9 carrying the S. cerevisiae SPT 15 gene in a pET expression vector was a gift of K. Arndt andF. Winston (Arndt et al., 1992). A modification of their purification scheme was developed and is presented below. BL21 (DE3) cells containing the plasmids pLysS and pKA9 are grown at 37 C in LB broth containing 25 pg/mL ampicillin and 30 pg/mL chloramphenicol. At a density of ß= 0.4, the broth is transferred to 30 C, TBP production is initiated by the addition of IPTG to 1 mM concentration and growth is continued at 30 C for 2 h. The cells are harvested and the cell pellets are stored at-70 C.All subsequent steps in the protocol are conducted at 4 C. Aliquots of the frozen cells are thawed on ice and resuspended (3 mL/g of cells) in 25 mM Hepes-KOH, 20% glycerol, 1 mM EDTA, 1 mM DTT, pH 7.9 (buffer A) with 100 mM KC1 containing aprotinin (1 pg/mL), chloromethyl ketone (20 pg/mL), leupeptin (1 wg/mL), trypsin inhibitor (100 pg/mL), and 100 mM PMSF (1 mM) protease inhibitors that are added to the buffer just prior to resuspension of the cells. The cells are lysed in a Biospec Bead-Beater using acid-washed dry glass beads (< 106 pm, Sigma) with an outer jacket filled with salt-saturated ice—water with four 30 s bursts, 3 min apart. The suspension is centrifuged in a Sorvall SS-34rotor at 18 000 rpm for 20 min. Chromosomal DNA is precipitated from the supernatant by slowly stirring in a protamine sulfate solution (6 mg/mL) to a final concentration of 0.3 mg/mL. The precipitate is removed by centrifugation as described above. A 2.2 x 25 cm column packed with 50 mL of Pharmacia S-Sepharose Fast Flow resin is equilibrated in buffer A with 100 mM KC1. Following sample loading, the column is washed with the equilibration buffer at 4 mL/min until A280 returns to baseline. The TBP is eluted with an 80 mL gradient of buffer A from 100 to 235 mM KC1 followed by a 240 mL gradient of 235—370 mM KC1; TBP elutes between 235 and 300 mM KC1. Column fractions are analyzed by SDS—PAGE; the TBP-containing fractions are pooled, concentrated to~ 1.5 mL using an Amicon Centricon 3 concentrator, and dialyzed overnight against buffer A with 100 mM KC1.