环式增强型绿色荧光蛋白热稳定性及Npu DnaE内含肽高反式剪接效率研究

摘要

目的 在体内借助Npu DnaE内含肽(intein)的高效反式剪接作用获得环式增强型绿色荧光蛋白(Cyc-EGFP).方法 对构建的融合表达载体pET/Npu DnaE intein/EGFP-28a( + )进行诱导、表达,之后利用亲和纯化获得高纯度的Cyc-EGFP和增强型绿色荧光蛋白(EGFP),并对二者的荧光强度和热稳定性进行比较.结果 Npu DnaE内含肽环化效率可达98%以上,明显优于Ssp DnaE内含肽,环化后的EGFP热稳定性提高了4～5℃.结论 Cyc-EGFP这一明显优势使蛋白环化在蛋白质工程等领域具有重要的应用价值.%Objective To produce Cyclic enhanced green fluorescent protein ( Cyc-EGFP) sre vivo using the highly efficient protein trans-splicing of Npu DnaE intein. Methods The fusion expression vector pET/Npu DnaE intein/EGFP-28a (+) was induced and expressed , and then using affinity purification high purity Cyc-EGFP and EGFP obtained , finally the fluorescence intensity and thermostability were compared. Results The trans-splicing activity of Npu DnaE intein had an efficiency above 98% , which was superior to that of Ssp DnaE intein. The Cyc-EGFP was 4 - 5^ more thermostable than the linear form. Conclusion The obvious advantage of Cyc -EGFP makes protein cyclization highly applicable to protein en - gineering and other fields.