一种通过GFP结合蛋白强化三分子荧光互补方法的建立

Establishment of a method for improving the tripartite split-GFP technique by applying GFP binding protein

[目的]将改造后的GFP(super-fold GFP,简写为sf GFP)作为三分子荧光互补技术的基础,通过应用多种GFP结合蛋白(GFP binding protein,GBP),筛选并验证可强化荧光互补作用的GFP结合蛋白。[方法]将sf GFP拆分为三部分(GFP1-9,GFP10和GFP11),建立GFP三分子荧光互补技术体系,利用多种GFP结合蛋白对GFP三分子荧光互补技术进行试验,通过观察荧光变化筛选可强化荧光互补作用的GFP结合蛋白。[结果]发现DARPin-GFP、GFP-enhancer和Nbsf GFP这三种GFP结合蛋白能够与GFP共定位并对GFP三分子重组装有增强效果,其中GFP-enhancer与Nbsf GFP增强效果约为原荧光强度4倍,DARPin-GFP约为2倍。[结论]GFP结合蛋白可作为一种新手段,使以GFP三分子荧光互补技术为基础的蛋白质-蛋白质相互作用研究的检测效率和灵敏度得到提高。

[ Objective] To discover and apply GFP binding protein（GBP） to research tripartite split - GFP technique based on modified GFP, super- folder GFP （sfGFP）can improve this technique. [ Methods ] Divide sfGFP into three parts, GFP1 -9, GFP10 and GFP11 ,and establish tripartite split- GFP method ,survey the green fluorescence on the GFP binding protein after they function on the reeombined split - GFP, then examine the advantageous GFP binding protein. [ Results ] DARPin - GFP, GFP - enhancer and NbsfGFP were selected as their different strengthening ability on recombination of split - GFP, GFP - en- hancer and NbsfGFP are around 4 times stronger than before, DARPin - GFP is almost twice. [ Conclusion ] GFP binding pro- tein acts as a new way which is capable of enhancing the renaturation and reinforce of split - GFP, and also can be used for pro- tein - protein interaction based on tripartite split - GFP technique by improving its efficiency and sensitivity.