抗GFP纳米抗体为亲和配基纯化缝隙连接蛋白26

Connexin26 purification by anti-GFP nanobody as affinity ligand

[目的]以表达纯化的抗GFP纳米抗体为亲和配基制作免疫亲和层析柱纯化缝隙连接蛋白26(Cx26),为其他与GFP融合表达的膜蛋白的纯化提供新的思路。[方法]用大肠杆菌表达镍柱纯化抗GFP纳米抗体,以其为亲和配基Sepharose 4B琼脂糖为亲和介质,制作抗GFP纳米抗体免疫亲和层析柱,并用其纯化Cx26-GFP融合蛋白。通过激光共聚焦显微镜和荧光尺寸排阻色谱法(FSEC)检测层析柱与Cx26-GFP融合蛋白的结合能力、稳定性;SDS-PAGE凝胶电泳、尺寸排阻色谱法(SEC)观察目的蛋白Cx26的纯度及均一性。[结果]抗GFP纳米抗体免疫亲和层析柱在激光共聚焦显微镜下呈现圆形、无色、直径约30~80μm的琼脂糖球,与Cx26-GFP融合蛋白结合后琼脂糖球发出绿色荧光。过柱前的溶膜后上清和过柱后的流穿液1经FSEC分析显示,两个样品均在洗脱体积11.5 mL时出现Cx26-GFP融合蛋白峰,流穿液1在此位置处的峰高比溶膜后上清降低了86%。柱上酶切过夜去除GFP标签后的流穿液2经SEC分析显示洗脱体积15 mL时出现左半峰高耸右半峰增宽的Cx26蛋白峰;峰尖处收集管液经SDS-PAGE凝胶电泳分析在25 kDa附近观察到Cx26蛋白条带。[结论]表达纯化的抗GFP纳米抗体有生物学活性,以此制备的抗GFP纳米抗体免疫亲和层析柱可稳定结合Cx26-GFP融合蛋白并纯化出杂质少、性状均一的Cx26蛋白。

[Objective]To make the immunoaffinity chromatography column that the affinity ligand was purified anti-GFP nanobody,and use this column to purify the Connexin26(Cx26),which provided a new idea for the purification of membrane proteins with GFP tag.[Method]The anti-GFP nanobody was expressed with E.coli and purified with Ni-NTA.With anti-GFP nanobody as affinity ligand and Sepharose 4B agarose as affinity medium,the anti-GFP nanobody immunoaffinity chromatography column was made.The binding ability and stability of this column with Cx26-CFP fusion protein were detected by laser confocal microscope and Fluorescence-detection Size-Exclusion Chromatography(FSEC).SDS-PACE and Size-Exclusion Chromatography(SEC)were used to observe the purity and homogeneity of the Cx26.[Result]The anti-GFP nanobody immunoaffinity chromatography column presented round,colorless with diameter of 30-80μm under the laser confocal microscope,which emitted green fluorescence when combined with Cx26-GFP fusion protein.The results of FSEC showed that the Cx26-GFP fusion protein peak appeared when the elution volume of both solubilization and flow through 1 in 11.5 mL,the peak height of Flow Through 1 at this position was 86%lower than solubilization.The Flow Through 2 was analysed by SEC and the results showed that the Cx26 protein peak with the left half peak rising and the right half peak widening which elution volume was 15 mL.SDS-PAGE analysis of the fraction by SEC in 15 mL showed a single electrophoretic band near 25 kDa which was Cx26.[Conclusion]The purified anti-GFP nanobody had biological activity,and the anti-GFP nanobody immunoaffinity chromatography column can stably bind to Cx26-GFP fusion protein,and the purify Cx26 protein with fewer impurities and uniform properties.