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[1] Cao Meng, Chang Weiwei, Xu Yang, Fang Linjing, et al. Construction and expression of GFP conjugated MIM-I-BAR [J]. Journal of Southeast University (English Edition), 2015, 31 (3): 353-357. [doi:10.3969/j.issn.1003-7985.2015.03.009]
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Construction and expression of GFP conjugated MIM-I-BAR()
绿色荧光蛋白探针标记的MIM-I-BAR基因构建和表达
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Journal of Southeast University (English Edition)[ISSN:1003-7985/CN:32-1325/N]

Volumn:
31
Issue:
2015 3
Page:
353-357
Research Field:
Biological Science and Medical Engineering
Publishing date:
2015-09-20

Info

Title:
Construction and expression of GFP conjugated MIM-I-BAR
绿色荧光蛋白探针标记的MIM-I-BAR基因构建和表达
Author(s):
Cao Meng1, Chang Weiwei1, Xu Yang2, Fang Linjing2, Liu Yuan2, Gu Ning1
1School of Biological Science and Medical Engineering, Southeast University, Nanjing 210009
2Chien-Shiung Wu College, Southeast University, Nanjing 210096
曹萌1, 常维维1, 许阳2, 方琳静2, 刘袁2, 顾宁1
1东南大学生物科学与医学工程学院, 南京 210009; 2东南大学吴健雄学院, 南京 210096
Keywords:
missing in metastasis;  inverse Bin-amphiphysin-Rvs;  green fluorescent protein;  plasmid;  expression;  purification
转移消失蛋白;  inverse Bin-amphiphysin-Rvs;  绿色荧光蛋白;  基重组因;  表达;  纯化
PACS:
Q816
DOI:
10.3969/j.issn.1003-7985.2015.03.009
Abstract:
To achieve a visible inverse Bin-amphiphysin-Rvs(I-BAR)domain recombinant of missing in metastasis(MIM)protein, the green fluorescent protein(GFP)encoding gene was cloned at the terminal of MIM-I-BAR as a probe. The DNA was successfully constructed on a 6xHis-tagged prokaryotic expression plasmid. The non-GFP labeled MIM-I-BAR encoding plasmid was also constructed as a control. Being successfully transformed into BL21(DE3)cells, the GFP-conjugated MIM-I-BAR(MIM-I-BAR-GFP)exhibits strong visible fluorescence, and the expression product can be easily detected by visual inspection, a fluorescence microscope, Western blot or ultraviolet and visible spectrophotometer. Moreover, examination of expression efficiency under various culture conditions revealed that the MIM-I-BAR-GFP gene has a high protein yield at 10 ℃, but not at the culture temperature of 37 ℃. This property is much different from that of the non-fluorescent MIM-I-BAR gene. This optimal expression condition is also proved to be feasible for protein production in midi-scale. The fluorescent recombinant MIM-I-BAR-GFP protein can serve as a useful tool in scientific research, biomedical application and pharmaceutical development.
为制备可视化的转移消失蛋白(MIM)的I-BAR结构域重组体, 克隆了顺联绿色荧光蛋白(GFP)探针编码序列的MIM-I-BAR基因.在6xHis标签原核表达质粒上成功构建了DNA序列.同时, 实现了未标记荧光探针基因的MIM-I-BAR质粒的构建以作实验对照.成功转染至BL21(DE3)大肠杆菌细胞后, GFP偶联的MIM-I-BAR(MIM-I-BAR-GFP)蛋白表现出很强的可视荧光, 该表达产物可方便的通过目测、荧光显微镜、免疫印迹和紫外可见分光光度计等多种手段进行检测.此外, 在考察不同条件下的蛋白表达效率过程中发现, 带有GFP探针的MIM-I-BAR重组蛋白在温度为10 ℃时产率最高, 而并非37 ℃.这一特征与非荧光标记的MIM-I-BAR明显不同.研究证实该最佳表达温度条件适用于重组蛋白产品中量制备.所开发的带有荧光探针的MIM-I-BAR蛋白产品及其制备工艺在科学研究、生物医学应用以及药物开发过程中均有较高的应用价值.

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Memo

Memo:
Biographies: Cao Meng(1983—), male, doctor; Gu Ning(corresponding author), male, doctor, professor, guning@seu.edu.cn.
Foundation items: The National Basic Research Program of China(973 Program)(No.2011CB933503), the National Natural Science Foundation of China for Key Project of International Cooperation(No.61420106012), China Postdoctoral Science Foundation(No.2013M541592).
Citation: Cao Meng, Chang Weiwei, Xu Yang, et al.Construction and expression of GFP conjugated MIM-I-BAR[J].Journal of Southeast University(English Edition), 2015, 31(3):353-357.[doi:10.3969/j.issn.1003-7985.2015.03.009]
Last Update: 2015-09-20