Expression and purification of polyhistidine-tagged firefly luciferase in insect cells - a potential alternative for process scale-up

The coleopteran firefly, Photinus pyralis, luciferase was produced in lepid opteran Trichoplusia ni insect cells using a baculovirus expression vector. The recombinant protein was equipped with a polyhistidine affinity tag at the carboxyl terminus and purified by immobilized metal-ion affinity chroma tography in combination with an expanded bed adsorption system. This approa ch enabled an efficient, one-step purification protocol of a genetically mo dified luciferase with properties similar to those of the authentic counter part. According to light emission measurements, the final yield of highly p urified protein was 23 mg l(-1) of cell culture. In addition, no specific i nteraction of interfering substances, such as, ATP, adenylate kinase, nucle oside diphosphokinase, as well as, creatine kinase of the final preparation were identified. Together, the results presented here dearly show that the baculovirus expression system in combination with immobilized metal-ion af finity chromatography is a potential strategy for process scale-up of polyh istidine tagged insect luciferase. (C) 2001 Elsevier Science B.V. All right s reserved.