title: In Situ Proteolysis for Crystallization of Membrane Bound Cytochrome P450 17A1 and 17A2 Proteins from Zebrafish.
abstract: Fish and human cytochrome P450 (P450) 17A1 catalyze both steroid 17α-hydroxylation and 17α,20-lyase reactions. Fish P450 17A2 catalyzes only 17α-hydroxylation. Both enzymes are microsomal-type P450s, integral membrane proteins that bind to the membrane through their N-terminal hydrophobic segment, the signal anchor sequence. The presence of this N-terminal region renders expression of full-length proteins challenging or impossible. For some proteins, variable truncation of the signal anchor sequence precludes expression or results in poor expression levels. To crystallize P450 17A1 and 17A2 in order to gain insight into their different activities, we used an alternative N-terminal sequence to boost expression together with in situ proteolysis. Key features of our approach to identify crystallizable P450 fragments were the use of an N-terminal leader sequence, a screen composed of 12 proteases to establish optimal cleavage, variations of protease concentration in combination with an SDS-PAGE assay, and analysis of the resulting fragments using Edman sequencing. Described in this unit are protocols for vector preparation, expression, purification, and in situ proteolytic crystallization of two membrane-bound P450 proteins.
authors.affiliation: Department of Biochemistry, Vanderbilt University, School of Medicine, Nashville, Tennessee.
authors.forename: Li
authors.initials: L
authors.lastname: Lei
authors.affiliation: Department of Biochemistry, Vanderbilt University, School of Medicine, Nashville, Tennessee.
authors.forename: Martin
authors.initials: M
authors.lastname: Egli
mesh_terms: D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D021382:Protein Sorting Signals; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013254:Steroid 17-alpha-Hydroxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D029961:Zebrafish Proteins; C000612994:cyp17a2 protein, zebrafish; D013254:Steroid 17-alpha-Hydroxylase; C000607915:cyp17a1 protein, zebrafish
keywords: crystallization; in situ proteolysis; membrane protein