云南农业大学学报(自然科学)
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云南农业大学学报(自然科学) 2017, Vol. 32 Issue (2) :191-196    DOI: 10.16211/j.issn.1004-390X(n).2017.02.001
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纳米荧光素酶的表达、纯化及性质鉴定
侯家鹏, 丁澦
复旦大学 生命科学学院, 上海 200438
Expression, Purification and Characterization of Nano Luciferase in Escherichia coli
HOU Jiapeng, DING Yu
School of Life Sciences, Fudan University, Shanghai 200438, China

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摘要 由荧光素酶(luciferase)催化的一大类发光反应被统称为生物发光(bioluminescence),该反应具有背景低、灵敏度高、线性范围广等特点,是高灵敏检测的发展方向之一。课题组在近期的研究中全基因合成了一种纳米荧光素酶(nano luciferase),研究清楚了其理化性质的基础上,将其作为一种新型的生物发光检测工具。将荧光素酶基因克隆至原核高表达载体上,通过自动诱导系统进行了表达。通过Ni-NTA亲和层析纯化和分子筛将重组纳米荧光素酶进行纯化,其产量可稳定达到每升菌25.3 mg,纯度超过98%;通过酶活实验鉴定了纳米荧光素酶的活性,在1.0×10-4~1.0×10-7 mg/mL的范围内,该酶均具有良好的线性反应,其线性检测下限低至1.0×10-7 mg/mL(约5 pmol)。纳米荧光素酶具有灵敏度高、数据重复性好、操作简便、稳定性高、易于保存等多重优点,可利用其作为基础平台,开发具有应用前景的超灵敏新型生物发光探针。
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侯家鹏
丁澦
关键词纳米荧光蛋白   原核表达   纯化     
Abstract: A large group of luminescence reactions catalyzed by luciferase are called bioluminescence. Since bioluminescence has been characterized as low background, high sensitivity and wide linear dynamic range. It is applied widely in sensitive molecular biology detection. In this research, we have synthesized a novel luciferase (nano luciferase) by whole gene synthesis, to make it a novel bioluminescent detection tool. Nano luciferase was cloned into a prokaryotic expression vector and expressed in an auto-induced system. Recombinant nano luciferase was purified by Ni-NTA affinity chromatography and size exclusive chromatography. The typical yield of recombinant nano luciferase was 25.3 mg from a liter culture medium with purity above 98%. Then, we tested the activity of recombinant nano luciferase, from 1.0×10-4 mg/mL to 1.0×10-7 mg/mL, nano luciferase has good linear response, the detection limit was as low as 1.0×10-7 mg/mL (around 5 pmol). The assay based on nano luciferase is ultrasensitive, good repeatable and easy operated. It would be applied as a basic tool to develop new sensitive bioluminescent probes with promising novel applications.
Keywordsnano luciferase (NLuc)   prokaryotic expression   purification     
Received 2017-01-24;
Fund:国家自然科学基金项目(31470764)。
Corresponding Authors: 丁澦(1978-),男,山东枣庄人,博士,副教授,主要从事结构生物学研究。E-mail:yuding@fudan.edu.cn     Email: yuding@fudan.edu.cn
About author: 侯家鹏(1991-),男,山东泰安人,硕士研究生,主要从事功能蛋白质克隆、表达与纯化研究。E-mail:houjiapeng11@163.com
引用本文:   
侯家鹏, 丁澦.纳米荧光素酶的表达、纯化及性质鉴定[J]  云南农业大学学报(自然科学), 2017,V32(2): 191-196
HOU Jiapeng, DING Yu.Expression, Purification and Characterization of Nano Luciferase in Escherichia coli[J]  Journal of Yunnan Agricultural University, 2017,V32(2): 191-196
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