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  

Thermostability of cyclic EGFP and highly efficient protein trans-splicing of Npu DnaE intein

作　　者：叶丙雨[1,2] 王政[2] 李德彬[2] 张乐之[2] 肖丽霞[2] 张部昌[1] 曹诚[2] 赵志虎[2] 

机构地区：[1]安徽大学生命科学学院,合肥230601 [2]军事医学科学院生物工程研究所,北京100071

出　　处：《军事医学》2012年第4期253-257,262,共6页Military Medical Sciences

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 NpuDnaE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）in 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 engineering and other fields.

关 键 词：蛋白环化 内含肽类 Cyc-EGFP 反式剪接 热稳定性 

分 类 号：Q78[生物学—分子生物学]