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HIGH‐PERFORMANCE LIQUID CHROMATOGRAPHY‐BASED PURIFICATION OF FIREFLY LUCIFERASES
Author(s) -
Branchini Bruce R.,
Rollins Cynthia B.
Publication year - 1989
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.1989.tb04326.x
Subject(s) - chromatography , luciferases , lampyridae , size exclusion chromatography , chemistry , sephadex , isoelectric focusing , high performance liquid chromatography , bioluminescence , luciferase , firefly protocol , biochemistry , biology , enzyme , zoology , transfection , gene
— A simple procedure is described for purifying luciferases from firefly lanterns in greater than 50% yield. The key step in the method is high‐performance liquid chromatography (HPLC) using a Bio‐Gel TSK DEAE‐5PW analytical (75 times 7.5 mm) anion exchange column. In separate experiments, partially purified protein solutions obtained from Photinus pyralis and Photinus macder‐motti lanterns by solubilization, ammonium sulfate fractionation and Sephadex G‐25 gel filtration were chromatographed. Luciferases from both species had relative molecular masses (M g ) equal to 61 ± 2 times 10 3 and were strongly reactive to anti‐P. pyralis luciferase antibody. Isoelectric focusing (IEF) experiments, HPLC retention times ( R t ), and bioluminescence emission spectra demonstrated that the luciferases from the different Photinus species were very similar, but not identical. The purification procedures described are most suitable for the isolation of 2–10 mg of protein; however, the use of a preparative column should enable the convenient isolation of larger amounts of protein. Also, this method should be readily adaptable to the isolation of luciferases from additional genera and species of fireflies.