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 lepidopteran 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 chromatography in combination with an expanded bed adsorption system. This approach enabled an efficient, one-step purification protocol of a genetically modified luciferase with properties similar to those of the authentic counterpart. According to light emission measurements, the final yield of highly purified protein was 23 mg l(-1) of cell culture. In addition, no specific interaction of interfering substances, such as, ATP, adenylate kinase, nucleoside diphosphokinase, as well as, creatine kinase of the final preparation were identified. Together, the results presented here clearly show that the baculovirus expression system in combination with immobilized metal-ion affinity chromatography is a potential strategy for process scale-up of polyhistidine tagged insect luciferase.
Michel
P
P
VTT Biotechnology and Food Research, FIN-02044 VTT, Espoo, Finland.
Torkkeli
T
T
Karp
M
M
Oker-Blom
C
C
D000818:Animals; D001517:Coleoptera / Q000201:enzymology / Q000235:genetics; D006639:Histidine / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D008156:Luciferases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D009036:Moths / Q000201:enzymology / Q000235:genetics; D010455:Peptides / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*
D010455:Peptides; D011993:Recombinant Fusion Proteins; C033223:polyhistidine; D006639:Histidine; D008156:Luciferases