Expression of soluble recombinant lipoxygenase from Pleurotus sapidus in Pichia pastoris

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• 作者：Sebastian Kelle ; Katerina Zelena ; Ulrich Krings ; Diana Linke ; Ralf G. Berger
• 关键词：Lipoxygenase ; Functional expression ; Kozak sequence ; Pleurotus sapidus ; Pichia pastoris
• 刊名：Protein Expression and Purification
• 出版年：March, 2014
• 年：2014
• 卷：95
• 期：Complete
• 页码：233-239
• 全文大小：915 K

文摘

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 伪-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 l^鈭? inducer. The recombinant enzyme showed an optimal lipoxygenase activity at pH 7 and 30-35 掳C and a v_max like the wild-type enzyme.