Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
8360721 | Protein Expression and Purification | 2014 | 7 Pages |
Abstract
The first heterologous expression of an iron-containing lipoxygenase from a basidiomycete in Pichia pastoris is reported. Five different expression constructs of the lipoxygenase gene LOX1 from Pleurotus sapidus were cloned and successfully transferred into P. pastoris SMD1168, but only one pPIC9K vector construct was functionally expressed. In this construct the vector-provided α-factor signal sequence was replaced by insertion of a second Kozak sequence between the signal sequence and the LOX1 gene. His+ transformants were screened for their level of resistance to geneticin (G418). Lox1 was expressed under different culture conditions and purified using the N-terminal His-tag. Relative enzyme activity increased significantly 48 h after methanol induction and was highest with 2 ml lâ1 inducer. The recombinant enzyme showed an optimal lipoxygenase activity at pH 7 and 30-35 °C and a vmax like the wild-type enzyme.
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Authors
Sebastian Kelle, Katerina Zelena, Ulrich Krings, Diana Linke, Ralf G. Berger,