Biochemical and Biophysical Research Communications
Regular ArticleExpression, Purification, and Characterization of Porcine Leukocyte 12-Lipoxygenase Produced in the Methylotrophic Yeast, Pichia pastoris
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Expression of soluble recombinant lipoxygenase from Pleurotus sapidus in Pichia pastoris
2014, Protein Expression and PurificationCitation Excerpt :Beside its advantage of simple genetic modification, easy cultivation and growth to high-cell densities, P. pastoris is able to perform eukaryotic post-translational modifications such as proteolytic processing, folding, disulfide bond formation and glycosylation [12,13]. Although heterologous expression of lipoxygenases from potato [14], tomato [15], pig [16], rat [17] and ascomycetes [18,19] have been described in P. pastoris, no functional expression of a iron-containing lipoxygenase from a basidiomycete in Pichia has become known. To learn more about the reasons for the numerous failures to functionally express basidiomycete genes in Pichia systems, the present study combined sequences coding for His-tagged and non-tagged proteins with and without the α-factor signal peptide.
Crystal structure of 12-Lipoxygenase catalytic-domain-inhibitor complex identifies a substrate-binding channel for catalysis
2012, StructureCitation Excerpt :Because the reticulocyte and leukocyte 12- and 15-lipoxygenases have closely related sequences and catalytic behavior, producing similar ratios of 12- and 15-hydroperoxides under comparable conditions, these isoenzymes are sometimes referred to as 12/15-lipoxygenases (Dobrian et al., 2011). The porcine leukocyte enzyme was first isolated and later cloned for expression in yeast, insect cells, and ultimately E. coli (Yokoyama et al., 1986; Reddy et al., 1994a, 1994b; Richards and Marnett, 1997; Rapp et al., 2009). A class of isoform-specific inhibitors was discovered for this 12-lipoxygenase, which led to a thorough kinetic analysis of the effect of 4-(2-oxapentadeca-4-yne)phenylpropanoic acid (OPP) on catalysis (Gorins et al., 1996; Richards, et al., 1999; Moody and Marnett, 2002).
Expression of manganese lipoxygenase in Pichia pastoris and site-directed mutagenesis of putative metal ligands
2005, Archives of Biochemistry and BiophysicsCitation Excerpt :This expression system has made it possible to analyze the putative manganese ligands by site-directed mutagenesis, and it may open the way for crystallization and X-ray analysis. Many plant and mammalian lipoxygenases have been successfully expressed in E. coli[9,15], and one mammalian lipoxygenase (porcine leukocyte arachidonate12-LO) in P. pastoris[35]. We also expressed Mn-LO in E. coli, but the protein lacked lipoxygenase activity.
Heterologous protein expression in the methylotrophic yeast Pichia pastoris
2000, FEMS Microbiology ReviewsLeukotriene A synthase activity of purified mouse skin arachidonate 8- lipoxygenase expressed in Escherichia coli
1999, Biochimica et Biophysica Acta - Molecular and Cell Biology of LipidsProfile of JTE-522 as a human cyclooxygenase-2 inhibitor
1998, Japanese Journal of Pharmacology