| 番茄Metacaspase基因(LeM)的克隆及其在大肠杆菌Rosetta中的高效表达 |
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| 引用本文： | 马秋敏,张雅丽,刘金娜,车英慧,谢寒丁,罗云波,曲桂芹.番茄Metacaspase基因(LeM)的克隆及其在大肠杆菌Rosetta中的高效表达[J].农业生物技术学报,2011,19(2). |
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| 作者姓名： | 马秋敏 张雅丽 刘金娜 车英慧 谢寒丁 罗云波 曲桂芹 |
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| 作者单位： | 中国农业大学食品科学与营养工程学院,食品生物技术实验室,北京100083 |
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| 基金项目： | 中央高校基本科研业务费专项资金，国家公益性行业(农业)科研专项项，国家自然科学基金 |
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| 摘 要： | 为获得可溶性的番茄Metacaspase蛋白,后续研究Metacaspase蛋白的酶学特性以及功能,利用RT-PCR方法克隆了番茄(Lycopersicon esculentum)中Metacaspase(LeM)全长cDNA,并成功地构建了LeM基因的原核表达载体重组质粒pET28a-LeM,转入大肠杆菌(Escherichia coli)BL21(DE3)PlysS和Rosetta中进行诱导表达,并对诱导表达条件进行了优化.SDS-PAGE结果显示,在37℃条件下,1 mmol/L IPTG诱导表达量最高,在BL21(DE3)PlysS菌株中重组蛋白主要以包涵体形式存在,而相同条件下在Rosetta菌株中多为可溶性表达.同时酶活测定显示,Rosetta上清中LeM的活性为33 RFU/min,显著高于BL21(DE3)PlysS 7RFU/min.本实验表明,不同菌株对Metacaspase的可溶性表达有很大影响.
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| 关 键 词： | 番茄 基因克隆 原核表达 |
Cloning of Lycopersicon esculentum Metacaspase Gene (Le M) and Its Effective Expression in Escherichia coli Rosetta |
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| Ma Qiumin,Zhang Yali,Liu Jinna,Che Yinghui,Xie Handing,Luo Yunbo,Qu Guiqin.Cloning of Lycopersicon esculentum Metacaspase Gene (Le M) and Its Effective Expression in Escherichia coli Rosetta[J].Journal of Agricultural Biotechnology,2011,19(2). |
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| Authors: | Ma Qiumin Zhang Yali Liu Jinna Che Yinghui Xie Handing Luo Yunbo Qu Guiqin |
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| Institution: | Ma Qiumin Zhang Yali Liu Jinna Che Yinghui Xie Handing Luo Yunbo Qu Guiqin Laboratory of Food Biotechnology,College of Food Science and Nutritional Engineering,China Agricultural University,Beijing 100083,China |
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| Abstract: | To obtain the fusion protein of Lycopersicon esculentum Metacaspase(LeM) and study the characteristics and function of it,we cloned the open reading frame of LeM in Lycopersicon esculentum by RT-PCR,and successfully constructed the prokaryotic expression vector pET28a-LeM for LeM expression.The recombinant plasmid pET28a-LeM was transformed into Escherichia coli BL21(DE3)PlysS and Rosetta for expression,induced by IPTG,and the conditions of the expression was optimized.SDS-PAGE revealed that the best expres... |
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| Keywords: | Metacaspase |
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