[1]唐玉林,米子岚,钟活权,等.利用SUMO技术表达可溶性的拟南芥AtRD22蛋白[J].深圳大学学报理工版,2015,32(6):610-616.[doi:10.3724/SP.J.1249.2015.06610]
　Tang Yulin,Mi Zilan,Zhong Huoquan,et al.Expressing the soluble AtRD22 protein of Arabidopsis thaliana in Escherichia coli using SUMO technology[J].Journal of Shenzhen University Science and Engineering,2015,32(6):610-616.[doi:10.3724/SP.J.1249.2015.06610]
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利用SUMO技术表达可溶性的拟南芥AtRD22蛋白()
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《深圳大学学报理工版》[ISSN:1000-2618/CN:44-1401/N]

卷:
第32卷
期数:
2015年第6期
页码:
610-616
栏目:
生物工程
出版日期:
2015-11-23

文章信息/Info

Title:
Expressing the soluble AtRD22 protein of Arabidopsis thaliana in Escherichia coli using SUMO technology
文章编号:
201506008
作者:
唐玉林1; 2; 米子岚1; 钟活权3; 江年琼1; 3
1）深圳大学生命与海洋科学学院，深圳 518060
2）深圳市海洋生物资源与生态环境重点实验室，深圳 518060
3）深圳市微生物基因工程重点实验室，深圳 518060
Author(s):
Tang Yulin1; 2;  Mi Zilan1;  Zhong Huoquan3;  and Jiang Nianqiong1; 2
1) College of Life and Marine Science, Shenzhen University, Shenzhen 518060, P.R.China
2) Shenzhen Key Laboratory of Marine Biological Resources and Ecological Environment, Shenzhen 518060, P.R.China
3) Shenzhen Key Laboratory of Genetic Engineering Microbes, Shenzhen 518060, P.R.China
关键词:
蛋白质工程; 拟南芥AtRD22; 融合标签; 小泛素相关修饰物; 基因克隆; 原核表达
Keywords:
protein engineering;  Arabidopsis AtRD22;  fusion tag;  small ubiquitin-related modifier (SUMO);  gene clone;  prokaryotic expression
分类号:
Q 943.2；Q 786
DOI:
10.3724/SP.J.1249.2015.06610
文献标志码:
A
摘要:
为了体外获得可溶性的拟南芥AtRD22蛋白，以拟南芥叶片提取的总核糖核酸（ribonucleic acid, RNA）为模版，反转录获得AtRD22的全长互补脱氧核糖核酸（complementary deoxyribonucleic acid, cDNA），分别构建AtRD22的原核重组表达载体pET32a-RD22和pSUMO-RD22，并转化大肠杆菌BL21（DE3）进行AtRD22蛋白的表达．在0.3 mmol/L的异丙基-β-D-硫代半乳糖苷（isopropyl-β-dithiogalactopyranoside, IPTG）诱导下，探索不同培养温度和诱导时间对可溶性蛋白表达的影响．结果表明，在实验条件下，转化pET32a-RD22的重组菌表达AtRD22蛋白的总量高于转化pSUMO-RD22的重组菌，但后者的可溶性AtRD22蛋白表达量明显高于前者．当诱导温度为28或16 ℃，时间分别为6.0或8.0 h以上时，转化pSUMO-RD22的重组菌中可溶性目的蛋白的表达量相对较大．为进一步在体外研究AtRD22蛋白质的结构和功能奠定了基础．
Abstract:
In order to obtain the soluble AtRD22 protein of Arabidopsis in vitro, the isolated total ribonucleic acid (RNA) of Arabidopsis thaliana leaves was used as the template for reverse transcription to amplify the full length complementary deoxyribonucleic acid (cDNA) of AtRD22. The obtained AtRD22 was constructed into two recombinant prokaryotic expression vectors pET32a-RD22 and pSUMO-RD22, respectively, and transformed into E.coli cells BL21(DE3) for the fusion protein expression. The induction temperature and time were optimized by a series of experiments conducted at 0.3 mmol/L isopropyl-Dithiogalactopyranoside (IPTG). Results show that the expression level of the total amount of the recombinant proteins in the pET32a-RD22-transformants is higher than that of pSUMO-RD22-transformants, but the level of soluble fusion protein in the latter is significantly higher than in the former. When the pSUMO-RD22-transformants are inducted for 6.0 h under 28 ℃ or for more than 8.0 h under 16 ℃, the expression level of the soluble fusion protein SUMO-RD22 is relatively higher. These results lay the foundation for the future study of the structure and function of AtRD22 in vitro.

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备注/Memo

备注/Memo:
Received：2015-07-23；Accepted：2015-09-24
Foundation：National Natural Science Foundation of China (30770184)； Research and Development Foundation of Science and Technology of Shenzhen (JCYJ20140724165855348)
Corresponding author：Associate professor Jiang Nianqiong. E-mali:jnq99@szu.edu.cn
Citation：Tang Yulin, Mi Zilan, Zhong Huoquan, et al. Expressing the soluble AtRD22 protein of Arabidopsis thaliana in Escherichia coli using SUMO technology［J］. Journal of Shenzhen University Science and Engineering, 2015, 32(6): 610-616.（in Chinese）
基金项目：国家自然科学基金资助项目（30770184）；深圳市科技计划资助项目（JCYJ20140724165855348）
作者简介：唐玉林（1968—），女（汉族），广西壮族自治区桂林市人，深圳大学副教授、博士．E-mail:yltang@szu.edu.cn
引文：唐玉林，米子岚，钟活权，等. 利用SUMO技术表达可溶性的拟南芥AtRD22蛋白［J］. 深圳大学学报理工版，2015，32（6）： 610-616.
更新日期/Last Update: 2015-11-06