Regular ArticleTruncated Human P450 2D6P: Expression in Escherichia coli, Ni2+-Chelate Affinity Purification, and Characterization of Solubility and Aggregation
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Membrane-attached mammalian cytochromes P450: An overview of the membrane's effects on structure, drug binding, and interactions with redox partners
2018, Journal of Inorganic BiochemistryCitation Excerpt :The first crystal structure of a mammalian CYP was published in 2000 and lacked the N-terminal transmembrane helix [90], which was deleted by enzyme engineering to facilitate crystallization. Most subsequent expression and crystallographic studies of mammalian CYPs have used truncated derivatives for the same reason [91–94]. It should be noted that these engineered structures are biochemically relevant because the deletions did not diminish the enzymes' catalytic activity [90] or eliminate their ability to attach to membrane surfaces [95].
Optimizations to achieve high-level expression of cytochrome P450 proteins using Escherichia coli expression systems
2013, Protein Expression and PurificationCitation Excerpt :An important tool used in heterologous expression of cytochrome P450 proteins is the modification of the N-terminal sequence. There exist two broad categories in N-terminal modification methods: (1) mutations that direct expression to the plasma membrane [22,24,26,29,33–35,42,44,45] and, (2) truncation/modification to redistribute protein localization into the cytosol [28,32,36,37,39,40,43]. Both approaches usually require further changes to increase expression levels and to obtain a catalytically active protein.
Characterization of hybrid bilayer membranes on silver electrodes as biocompatible SERS substrates to study membrane-protein interactions
2010, Colloids and Surfaces B: BiointerfacesFunctional expression of N-terminally tagged membrane bound cytochrome P450
2009, Protein Expression and PurificationExpression of human cytochrome P450 46A1 in Escherichia coli: Effects of N- and C-terminal modifications
2004, Archives of Biochemistry and BiophysicsCitation Excerpt :The four mammalian P450s (2C5, 2B4, 2C8, and 2C9), whose tertiary structures have been solved by the X-ray analysis and published, have the C-terminal His-tag; however, their N-terminal amino acid sequences, MAKKTSSKG(R/K), have four positively charged amino acid residues [12,13,21,22]. We are aware of expression of only two N-terminally His-tagged truncated P450s, 2D6 and 2C2 [23,24]. In both cases, the engineered enzyme was either 75% (2D6) or 50% (2C2) cytosolic.
Protein engineering of thromboxane synthase: Conversion of membrane-bound to soluble form
2003, Archives of Biochemistry and BiophysicsCitation Excerpt :Quite similar properties with respect to oligomeric state and subcellular distribution were observed in the present study for TXAS/2C5 in which helix F and the F–G loop of TXAS were substituted by the corresponding P450 2C5/3LVdH. Several groups have attempted to convert Family 2 P450s to soluble forms using protein engineering techniques [33–35]. These recombinant proteins either exhibited polydispersive states or tended to aggregate unless detergent was included throughout purification, thus making crystal growth difficult.