亮氨酸拉链结构蛋白在大肠杆菌重组子中的表达(英文)  

Study on Expression Condition of a Leucine-Zipper Protein in the Recombinant Escherichia coli

作　　者：何冬兰[1] 牧野圭祐 

机构地区：[1]中南民族大学化学与生命科学学院,武汉430074

出　　处：《中南民族大学学报（自然科学版）》2002年第3期4-7,共4页Journal of South-Central University for Nationalities：Natural Science Edition

基　　金：国家留学基金委员会资助项目 (9984 2 135 )

摘　　要：酵母转录因子 GCN4是通过亮氨酸拉链 (b ZIP)结构结合 DNA的蛋白质之一 ,当 GCN 4二聚体与 DNA结合时 ,亮氨酸拉链区的 2个单体结合为平行的卷曲螺旋结构 ,而其基区由无规线团结构变为α螺旋 .为探讨亮氨酸拉链蛋白与 DNA的结合机理 ,设计了含有 GCN4亮氨酸拉链蛋白基区结合 DNA的必需氨基酸的折叠片段 ,并将其克隆到 Escherichia coli BL 2 1,讨论了此亮氨酸拉链蛋白的表达条件 .在蛋白质的小量表达试验中 ,重组子 Es-cherichia coli BL 2 1于 5 m L含有 5 0μg/m L氨苄青霉素和 34μg/m L氯霉素的 L B液体培养基中培养至对数期 ,加入不同浓度的 IPTG,继续培养以诱导蛋白质的表达 ,在不同的时间 (如 :诱导前 ,诱导 2 ,4 ,6 ,8h)取样 10 0μL到1.5 m L离心管中、离心收集沉淀 ,将沉淀悬浮于样品缓冲液中 ,用 10 % SDS- PAGE检测 ;在 10 L含氨苄青霉素和氯霉素的 L B液体培养基中进行了大量表达 ,根据小量表达的试验结果确定了 IPTG的浓度和诱导时间 .结果表明 :含有这种拉链蛋白质的重组子 Escherichia coli BL 2 1在 37℃下小量培养时 ,0 .1～ 0 .8mm ol的 IPTG均可在 2～ 10 h内诱导该蛋白质表达 ;而大量培养时 ,0 .2 m mol和 0 .4 mm ol的 IPTG在 37℃均不可能诱导表达 ,只在2 8℃时才表达 ;The yeast transcriptional factor GCN4 is one of a large family of DNA binding proteins identified by the basic leucine zipper structural motif. When the GCN4 dimer binds a specific DNA sequence, the leucine zipper region of two monomers is assembled into a parallel coiled coil, whereas structure of the basic region transits from the random coil to an α helix. To detect the mechanism of bZIP binding DNA, we designed a small folding domain containing the amino acids necessary for DNA binding of the basic leusine zipper protein GCN4 and cloned it into Escherichia coli BL21. The expression condition of the protein was discussed in this research. In the small scale expression of the protein, the recombinant Escherichia coli BL21 is incubated in 5 mL LB liquid medium including 50 μg/mL ampicillin and 34 μg/mL chloramphenicol till it get to exponential phase. Induce the culture by adding IPTG at different concentration and continue incubation. At various time points during the induction period (e g 0, 2, 4, 6, 8 h), take 100 μL of the culture to 1.5 mL centrifuge tube and centrifuge it and get the pellet. Suspend the depositions with loading buffer. Check them with 10% SDS PAGE. The large scale expression of the protein is done with 10 L LB liquid medium including ampicillin and chloramphenicolat the best induction time and inducer concentration gotten from the small scale expression. The result shows the leucine zipper protein is produced in Echerichia coli BL21 induced by 0.1~0.8 mmol IPTG for 2~10 h at 37℃ after the OD600 value to 0.4~0.6 in small scale expression. But it can't express at 37℃ in large scale expression at the above condition. The protein expresses successfully at 28℃.The best induced time is 4~6 h and inducer concentration is 0.2 mmol in the small scale expression as well as in the large scale expression. The temperature for large scale expression is 28℃ not 37℃.

关 键 词：亮氨酸拉链结构 诱导 蛋白质 大肠杆菌 酵母转录因子 亮氨酸拉链蛋白 DNA 重组子 

分 类 号：Q784[生物学—分子生物学] R378.21[医药卫生—病原生物学]