Construction of Luciferase Expression Vector and Its Expression in Pichia pastoris
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Abstract:
Firefly Luciferase (Fireflyluciferase, FL) is the core component of ATP bioluminescence assay, which plays a critical role in the detection of foodborne microorganisms in food industrythrough the correlation between the luminous intensity and ATP concentrations ., In order to realize the heterologous expression of luciferase in recombinant P. pastoris GS115, the luciferase gene was amplified and cloned into the eukaryotic expression vector pPIC9K. And then the vector was linearized and was transformed into the strain GS115 by electricity to screen the positive transformants. Later ATP bioluminescence assay was employed to test the enzyme activity of both extracellular and intracellular crude enzyme liquid following the expression induced by methanol. The crude enzyme was then purified by ultrafiltration, anion exchange and size exclusion chromatography sequentially. The extracellular and intracellular crude enzyme liquid all had relatively higher enzyme activity after a 96 h-methanol induction, which were 1.45×106 RLU/mL and 1.58×109 RLU/mL, respectively. SDS-PAGE and westernblot analysis indicated that the protein size of FL was about 70 ku. The purified luciferase activity was 7.0×108 RLU/mg with the purification fold of 19.3 and the yield of 48 mg/L. In conclusion, our study here demonstrated that the firefly luciferase could be well expressed and purified in the eukaryotic expression system as P. pastoris GS115.
