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Updated: Jul 1, 2026
Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli
Published on: January 6, 2015
[Prokaryotic expression, purification and identification of VEGFR2 D3.4/GST fusion protein in E.coli]
Yu-ying Wei1, Chao-jun Song, Yuan-jie Sun
1Department of Immunology, Fourth Military Medical University, Xi'an 710032, China.
Aim:
To construct a prokaryotic expression vector for the expression of VEGFR2 D3.4/GST fusion protein, Express and purify the fusion protein.
Methods:
The coding sequence of the third and fourth extracellular domain of human VEGFR2 gene fragment was synthesized and subcloned into pGEX4T-1 vector downstream of the GST fragment, an E.coli expression vector, to construct a recombinant plasmid pGEX4T-VEGFR D3.4. Then the plasmid was transformed into E.coli BL21 (DE3) pLysS and induced to express fusion protein VEGFR2 D3.4/GST with IPTG. The expressed protein was purified by washing in urea and detected by SDS-PAGE and Western blot.
Results:
SDS-PAGE analysis showed that a novel protein with the expected molecular mass (M(r);) about 46 000 was expressed with the inducement of IPTG. And it existed mostly in the form of inclusion body. Grayscale scanning showed that the expressed VEGFR2 D3.4/GST fusion protein accounted for 38.6% of the total bacterium protein. After the purified product was washed by urea, its purity reached 87.1%. Western blot confirmed the recombinant protein was VEGFR2 D3.4/GST fusion protein.
Conclusion:
High purification VEGFR2 D3.4/GST fusion protein is obtained through the E.coli expression system.
