研究了Lg-Flo1蛋白N端的诱导表达条件及与甘露糖的结合活性。将构建的巴氏酵母Lg-FLO1基因N端序列的重组表达载体pETFL1进行双酶切验证,转化大肠杆菌BL21(DE3),通过菌落PCR鉴定阳性转化子,并进行诱导表达,利用SDS-PAGE检测不同诱导条件对目的蛋白N-Lg-Flo1表达的影响,并对目的蛋白进行变性溶解、纯化、复性及活性测定。结果表明,酶切证实重组表达载体含有N-Lg-FLO1基因片段;在LB培养基中的表达量高于TB培养基;和IPTG相比,乳糖诱导同样可以表达含量较高的目的蛋白且成本较低。以乳糖作为诱导剂,终质量浓度为0.2 g/L,诱导温度37 ℃,诱导6 h,目的蛋白的表达量均高于30%;荧光光谱分析表明,复性后的N-Lg-Flo1蛋白具有与甘露糖结合的活性。该研究为大规模生产絮凝蛋白并以此为原料制备糖的特异性吸附剂奠定了基础。
The induction condition and binding activity of N-terminal of Lg-Flo1 protein with mannose were studied. The recombinant expression vector pETFL1 with N-terminal sequence of Lg-FLO1 gene from Pasteurella pastoris was digested, identified and transformed into Escherichia. coli BL21 (DE3). The positive transformants were identified and Lg-FLO1 gene was expressed and the effects of induction condition on expressing target protein were studied, followed by determining the activity of target protein. The expression level of N-lg-Flo1 protein in LB medium was higher than that in TB medium. Compared with IPTG induction, the target protein was expressed at a higher level with lactose. The highest expression level of the target protein was obtained after 6 h induction with 0.2 g/L lactose at 37℃. Besides, the fluorescence spectrum analysis revealed mannose-binding activity of the renatured N-Lg-Flo1 protein. This research establishes a base for large-scale production of flocculant proteins that can be used as raw materials to prepare specific adsorbents for carbohydrates.
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