[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()

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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

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

Keywords:

missing in metastasis;  inverse Bin-amphiphysin-Rvs;  green fluorescent protein;  plasmid;  expression;  purification

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.

References:

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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]

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