بيان ژن و تخليص آنزيم لوسيفراز و سنجش ATP سلولي

Expression and purification of the luciferase enzyme and in vivo ATP Assay

Introduction: In this study, gene expression and purification of luciferases from the firefly, Lampyris turkestanicus, and optimization of cellular ATP measurements were performed. Methods: cDNA encoding luciferases from Lampyris turkestanicus was transferred from pQE30 vector into pET28a expression vector and pLtu28 was built. Newly constructed vector was expressed in E. coli XL1 Blue and the recombinant luciferase was purified using Ni-NTA Sepharose column. Enzymatic properties (Km and Vmax) for ATP were measured using luminescence assay. Standard curve of ATP was obtained by Promega ATP detection kit and the designed method based on the L. turkestanicus luciferase and ATP serial dilution. Moreover, bacterial ATP was measured by Promega kit and the designed method using L. turkestanicus luciferase. Results: Results showed that ligation of L. turkestanicus luciferase encoding cDNA into pET28a and transformation of competent cells induced by the recombinant vector was performed efficiently. Using luciferin, positive colonies were screened and cultured. SDS-PAGE showed that recombinant luciferase was efficiently purified by Ni-NTA Sepharose column. ATP standard curve and measurement of bacteria, using Promega and the designed method by L. turkestanicus luciferases showed high similarities. Conclusion: A method based on L. turkestanicus luciferases was designed. Comparison of the developed assay with promega kits in identification of bacterial concentration show its high quality and potent ability in ATP detection.