中南民族大学学报（自然科学版）2002，Vol.21Issue(3)：4-7,4.
亮氨酸拉链结构蛋白在大肠杆菌重组子中的表达
Study on Expression Condition of a Leucine-Zipper Protein in the Recombinant Escherichia coli
摘要
Abstract
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/表达/诱导Key words
leucine zipper protein/expression/induce分类
生物科学引用本文复制引用
何冬兰,牧野圭祐..亮氨酸拉链结构蛋白在大肠杆菌重组子中的表达[J].中南民族大学学报（自然科学版）,2002,21(3):4-7,4.基金项目
国家留学基金委员会资助项目(99842135) （99842135）
