穿膜肽标记的荧光素酶蛋白在大肠杆菌中表达条件的优化及其活性研究

Optimization of expression conditions of luciferase protein labeled by membrane peptide in Escherichia coli and study of its activity

将重组质粒转化到大肠杆菌BL21(DE3)中,在不同的诱导时机、诱导温度、IPTG的浓度和Mg^(2+)浓度条件下培养大肠杆菌,通过SDS-PAGE和发光强度检测分析TAT-LUC的酶活性强度及表达量;将高活性的TAT-LUC与LUC对比检测ATP,通过发光强度分析TAT-LUC的敏感性和可靠性。表明:在含Mg^(2+)浓度为50mmol/L的LB培养基中将大肠杆菌培养至OD600为0.6时,加入终浓度为0.5mmol/L的IPTG,22℃诱导16h可获得大量高活性的TAT-LUC;相比LUC,TAT-LUC可以检测到小于10nmol/L的ATP,发光强度与ATP含量呈强相关性(R2=0.99);并且不用裂解细胞,TAT-LUC可直接检测至少40个细胞内ATP;穿膜肽标记的荧光素酶蛋白成功用于检测活细胞内的ATP含量。

The recombinant plasmid is transformed into Escherichia coli BL21（DE3）,and Escherichia coli is cultivated under the conditions of different induction time,induction temperature,concentration of IPTG and Mg（2＋） concentration.Through SDS-PAGE and luminescence intensity detection,the enzyme activity intensity and expression of TAT-LUC are analyzed.The high active TAT-LUC is compared with LUC to detect ATP,and through the luminous intensity,the sensitivity and reliability of TAT-LUC are analyzed.The results show that when the Escherichia coli in an LB medium with the concentration of Mg（2＋） which is 50 mmol/L is cultivated from OD600 to 0.6,IPTG with a final concentration of0.5 mmol/L is added and a large amount of the highly active TAT-LUC can be obtained after the 16 hinduction at 22℃.In comparison with LUC,TAT-LUC can detect ATP of less than 10 nmol/L,and the intensity of luminescence is strongly correlated with the content of ATP（R2=0.99）.Without cracking cells,TAT-LUC can directly detect at least40 intracellular ATPs.The luciferase protein labeled by membrane peptide has been successfully used to detect the content of ATP in living cells.