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  • Title: [Prokaryotic expression, purification and identification of VEGFR2 D3.4/GST fusion protein in E.coli].
    Author: Wei YY, Song CJ, Sun YJ, Gong JY, Jin BQ, Yang AG, Yang K.
    Journal: Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi; 2008 Sep; 24(9):870-2. PubMed ID: 18782519.
    Abstract:
    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.
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