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  • Title: [Expression and purification of human sucrase protein in E.coli].
    Author: Shao M, Wang X, Zhang Q, Niu X, Wang Y, Zhou H, Ge Z.
    Journal: Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi; 2015 Dec; 31(12):1629-32. PubMed ID: 26648296.
    Abstract:
    OBJECTIVE: To construct prokaryotic expression vector pET-28a(+)-human sucrase (hSUC) and express hSUC fusion protein in E.coli. METHODS: The hSUC gene fragment was amplified by reverse transciption PCR (RT-PCR) and cloned into pET-28a(+) vector to construct the prokaryotic expression vector pET-28a(+)-hSUC. The recombinant plasmid was then transformed into E.coli BL21. Hisdidine (His)-tagged fusion proteins were induced by isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified by nitrilotriacetic acid (Ni-NTA) agarose resin. The purified fusion proteins were identified by SDS-PAGE and Western blotting. RESULTS: RT-PCR showed that sub-clone of hSUC was about 1482 bp. The recombinant plasmid was correctly constructed as demonstrated by sequencing and restriction enzyme analysis. The molecular mass of the fusion protein was about 61 240. Western blotting showed that the fusion proteins bound specifically to hSUC antibody. CONCLUSION: The hSUC protein has been successfully expressed and purified in E.coli.
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