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Logo 22nd ISBC & 20th ISLS


Analytical, clinical and medical applications of luminescence

Engineering of firefly luciferase for cell line-based applications

Chadaporn Kantiwiriyawanitch1, Vinutsada Pongsupasa1, Pratchaya W.1, Jittima P.1, Ubolsree L.2, Ruchanok T.3, Thanyaporn W.1, Yoshihiro N.4, Yoshihiro O.5, Pimchai C.1

1School of Biomolecular Science and Engineering, Vidyasirimedhi Institute of Science and Technology (VISTEC), Wangchan Valley, Rayong, 21210 (Thailand)., 2National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Agency (NSTDA), Pathum Thani, 12120 (Thailand)., 3Department of Biochemistry and Center for Excellence in Protein and Enzyme Technology, Faculty of Science, Mahidol University, Bangkok, 10400 (Thailand)., 4National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu, Kagawa 761-0395, Japan., 5Osaka Institute of Technology (OIT), Osaka, Osaka 535-8585, Japan

E-mail: chadaporn.k_s20@vistec.ac.th

Firefly luciferase (Fluc) is widely used as bioreporter genes for monitoring gene expression or studying cellular signaling in living cells or organisms. However, Fluc is not very stable upon a long-term storage. In this work, we employed rational and computer-assisted protein engineering approach to enhance Fluc stability. We successfully generated two thermostable variants of Fluc, exhibiting enhanced half-lives (t ½) at 37 oC. Specifically, for VISTEC1, the improvement is 3.5-fold compared to the native enzymes, while for VISTEC, it is 2.8-fold. The two variants which showed superior biochemical properties than the wild-type enzyme could be expressed in HEK293T and exhibited greater light signals than that of the native enzyme, up to 4.2-fold compared to the native Fluc. Furthermore, we also demonstrated the use of engineered Fluc as cell-line bioreporter applications to measure inhibitors and activators activities of the cell signalling pathways. Therefore, our results indicate that these variants with improved properties are suitable for applications as reporter genes in mammalian cells.

Keywords: Firefly luciferase; Thermostability; Protein engineering; Reporter genes; Cell signaling

Acknowledgments: This work was supported by a grant from VISTEC and NSTDA (to P. C., U. L., V. P. and C.K.). We thank Frontier Research Center (FRC) of VISTEC for instrumental facilities.

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