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Optimizing Expression in E.coli and Bioactivity Assay of Recombinant GST-gAD

Author: WangJing
Tutor: LuoZhiJun；YinDong
School: Nanchang University
Course: Biochemistry and Molecular Biology
Keywords: Adiponectin Globular domain Expression of recombinant protein Breast carcinoma cell line
CLC: Q78
Type: Master's thesis
Year: 2008
Downloads: 89
Quote: 0
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Abstract


Objective: To explore the best expressing condition of the recombinant GST-gAD( globular domain of human adiponection,gAD) in E.coli and study its bioactivity in inhibiting the proliferation of human breast cancer cell MDA-MB-231.Methods:①Competent bacterium JM109 was prepared with CaCl2. The plasmid pGEX-KG-gAD and the empty plasmid pGEX-KG, as control, were transformed into E.coli host JM109;②The best expressing condition of GST-gAD was explored through orthogonal design which took L 9(34),including: growth temperature, IPTG concentration,culture density,incubation time as indices by orthogonal design assistantⅡ;③GST- gAD and GST were purified by GSH-agarose;④The effect of GST—gAD on proliferation of MDA-MB-231 cells were measured by MTT essay.Results:①The pGEX-KG-gAD recombinant vector was transformed successfully into E.coli host JM109;②The expression level was highest in the conditions of OD600 value 1.0,1.0mmol/L IPTG, 32℃and 1.5 hours of induction time. The amount of the fusion protein was about 6% of the total bacterial protein;③The molecular weight of GST-gAD was 42KD;④The GST-gAD can obviously inhibit the proliferation of MDA-MB-231 when its concentration was more than 0.5μmol/L. Conclusion:①The best expressing condition of the recombinant GST-gAD in Ecoli was determined through orthogonal design;②The GST-gAD fusion protein was efficiently expressed in E.coli and showed natural biological activities.

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