GST和His标记的重组蛋白制备及吸附纳米ZnO的研究
Preparation of Recombinant Protein with GST and His Tag and Its ZnO-Binding Quality
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摘要: 以pET-28a(+)载体为模板人工设计引物,通过PCR反应扩增带有质粒上游6聚组氨酸(6×Histidine)序列的目的基因片段,克隆到原核表达载体pGEX-4T-3中,筛选及鉴定阳性重组子pGEX-His,转化<em<E.coli</em< BL21细胞后通过IPTG诱导表达. 由谷胱甘肽(GST)和6聚组氨酸标记的重组蛋白GST-His得到表达,利用Ni-NTA系统进行纯化. 通过SDS-PAGE电泳和Western blot 分析鉴定,由LC-MS/MS质谱分析确定所得蛋白为目标产物. 通过蛋白结Abstract: The plasmid pET-28a(+) was used as templates to PCR-amplify objective fragment containing the upstream 6×Histidine sequence with designed primers. PCR products were ligated into a prokaryotic expression vector pGEX-4T-3. Positive <em<E.coli</em< clones containing recombinant plasmid pGEX-His were selected and identified, followed by transformation into <em<E.coli</em< BL21 cells and induction with IPTG. The recombinant protein GST-His was expressed with GST and His tag, purified with Ni-NTA system, identified by SDS-PAGE electrophoresis and Western blot analysis and confirmed by LC-MS/MS analysis. Affinity adsorption test on the proteins binding with nanoparticles demonstrated that the fusion protein had a specific adsorption with ZnO nanoparticles.
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