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[目的]获得可用于蛋白晶体筛选的高纯度Bnip3蛋白。[方法]将Bnip3截断基因构建到原核表达载体上并转化大肠杆菌。IPTG诱导表达后通过GST柱纯化目标蛋白。TEV酶切除GST标签后采用凝胶过滤层析纯化Bnip3蛋白。[结果]Bnip3截断基因重组体成功构建并诱导表达目标蛋白。通过GST亲和层析得到可溶性的携带GST标签的Bnip3(1~111)和Bnip3(1~152)蛋白。切除GST标签的Bnip3(1~152)通过凝胶过滤层析得到构象均一的二聚体蛋白。[结论]采用原核表达、亲和层析和凝胶过滤层析可获得构象均一的高纯度二聚体Bnip3蛋白。
[Objective] The aim was to obtain high-purity Bnip3 protein which can be used for protein crystal screening. [Method] The Bnip3 truncated gene was constructed into prokaryotic expression vector and transformed into E. coli. After IPTG induced expression, the target protein was purified by GST column. After the TEV enzyme excised the GST tag, the Bnip3 protein was purified by gel filtration chromatography. [Result] The Bnip3 truncated gene recombinant was successfully constructed and induced to express the target protein. Soluble GST-tagged Bnip3 (1 ~ 111) and Bnip3 (1 ~ 152) proteins were obtained by GST affinity chromatography. Gn-tagged Bnip3 (1 to 152) was gel-chromatographed to give a conformationally homogeneous dimer protein. [Conclusion] Homomorphic high purity dimer Bnip3 protein was obtained by prokaryotic expression, affinity chromatography and gel filtration chromatography.