A convenient affinity chromatography-based purification of firefly luciferase

A method is described for purifying luciferase from firefly lanterns in very good yield. The enzyme was extracted from Photinus lanterns, dialyzed, and further purified by affinity chromatography with 6-aminohexanoic acid-Sepharose 4b benzylamine. Fractions containing luciferase activity were pooled, concentrated by ultrafiltration, and crystallized by dialysis against a low-ionic-strength buffer. The crystalline enzyme appears to be homogeneous by sodium dodecyl sulfate-gel electrophoresis. The entire protocol described was conveniently performed in less than 3 days and yielded 6.2 mg of enzyme from 2.2 g of firefly lanterns. Furthermore, the affinity column employed can be reused at least five times without appreciable loss of binding capacity. Luciferase was chromatographed with several other affinity gels and the results are compared. It appears that 6-aminohexanoic acid-Sepharose 4b benzylamine does not function as a true bioaffinity gel, but rather as a hydrophobic interaction support.