In Situ Proteolysis for Crystallization of Membrane Bound Cytochrome P450 17A1 and 17A2 Proteins from Zebrafish

Li Lei1, Martin Egli1

1 Department of Biochemistry, Vanderbilt University, School of Medicine, Nashville, Tennessee
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 29.16
DOI:  10.1002/0471140864.ps2916s84
Online Posting Date:  April, 2016
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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. © 2016 by John Wiley & Sons, Inc.

Keywords: in situ proteolysis; crystallization; membrane protein

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Vector Preparation, Expression, Purification, and In Situ Proteolytic Crystallization Of Membrane‐Bound P450 Proteins
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Vector Preparation, Expression, Purification, and In Situ Proteolytic Crystallization Of Membrane‐Bound P450 Proteins

  • Zebrafish
  • Plasmid vectors: pET 17b expression vectors (Novagen)
  • Qiagen One‐Step RT‐PCR kit
  • E. coli DH5 cells for cloning (Vanderbilt University Core Lab)
  • BL21 (DE3) Gold (or plysS) for overexpressing protein (Invitrogen)
  • LB agar plates
  • Antibiotics: e.g., Kanamycin sulfate (Research Products International, cat. no. K22000‐5.0), Ampicillin sodium salt (Research Products International, cat. no. A40040‐5.0)
  • LB broth (see recipe)
  • δ‐Aminolevulinic acid hydrochloride (ALA; CHEM‐IMPEX International, cat. no. 01433)
  • L‐Arabinose (Gold Bio Technology, CAS No. 87‐72‐9)
  • IPTG (Research Products International, cat. no. I56000‐5.0)
  • Tris
  • TES buffer
  • Lysozyme
  • Buffer A (see recipe for buffers for chromatography)
  • Ice
  • Ni‐NTA Agarose resin (Qiagen, cat. no. 30250)
  • Imidazole (Sigma, cat. no. I2399‐500 G)
  • Buffer B (see recipe for buffers for chromatography)
  • Liquid nitrogen
  • Sepharose
  • Deionized water
  • Buffer C (see recipe)
  • Sodium chloride (NaCl)
  • CMC detergent (see recipe)
  • C12E9 Polyoxyethylene(9)dodecyl Ether • Thesit • Polydocanol • α‐Dodecyl‐w‐Hydroxy‐Poly(oxy‐1,2‐Ethanediyl) (Anatrace, cat. no. Apo‐12950X1ml)
  • Buffer D (see recipe)
  • For SDS‐PAGE: mini‐protean TGX precast 4% to 20% (w/v) Tris‐glycine gel, (BIO‐RAD, cat. no 601‐1105)
  • Coomassie Brilliant Blue R 250 (MP Biomedical, cat. no. 04821616)
  • Pregnenolone (Steraloids), 17α‐OH pregnenolone (Steraloids), and Abiraterone (Selleckchem)
  • Ethanol
  • Dimethyl sulfoxide (DMSO)
  • Phenylmethanesulfonyl fluoride (PMSF; Sigma, cat. no. P7626‐25 G)
  • Laemmli Sample buffer for SDS‐PAGE (Bio‐Rad, cat. no. 161‐0737)
  • SDS gel stain (see recipe)
  • SDS gel detain solution (see recipe)
  • Crystallization screens:
  • Index kit (Hampton Research, cat. no. HR2‐ 144)
  • Crystal screen (Hampton Research, cat. no. HR2‐110)
  • Crystal screen 2 (Hampton Research, cat. no. HR2‐112)
  • PEG/Ion screen (Hampton Research, cat. no. HR2‐126)
  • Trypsin
  • Seed bead (Hampton Research, cat. no. 2‐320)
  • Methanol (Fisher Scientific, cat. no. A433P‐4)
  • 3‐Cyclohexylamino‐1‐propanesulfonic acid (CAPS buffer; see recipe)
  • K 2HPO 4 and KH 2PO 4 (Research Products International, cat. no. 41300‐500 and 41200‐500, respectively)
  • Detergent Screen HT (Hampton Research, cat. no. HR2‐406)
  • Proti‐Ace Kit (Hampton Research, cat. no. HR2‐429)
  • Proti‐Ace 2 Kit (Hampton Research, cat. no. HR2‐432)
  • 37°C incubator
  • 250‐ml and 2.8‐liter flasks
  • Incubator shaker
  • Spectrophotometer (Shimadzu Scientific, cat. no. UV‐2401PC)
  • Beckman GS6 KR centrifuge (Beckman Instrument, cat. no. 362134)
  • 50‐ml tubes
  • Sonicator
  • Protean software package (‐protean.aspx)
  • 1.5‐ml microcentrifuge tubes
  • CrystalQuick 96‐well Greiner 609191 (Hampton Research, cat. no. HR3‐192)
  • Microwave oven
  • Mosquito crystal liquid handler (TTP labtech, cat. no. Mosquito)
  • Rock Imager (Formulatrix cat. no., rock imager 1000)
  • Vortex mixer
  • Invitrolon PVDF filter paper sandwich, 0.45‐μm pore size (Invitrogen, cat. no. LC2005)
  • MRC 2 well crystallization plates (Hampton Research, cat no. HR3‐082)
  • Amicon ultra centrifugal filters, 30 K (Merck Millipore, cat. no. UFC 903024)
  • Q sepharose fast flow (GE Healthcare, cat. no. 17‐0510‐01)
  • SP sepharose fast flow (GE Healthcare, cat. no. 17‐0720‐01)
  • Size‐exclusion columns
  • Superdex 75 10/30 GL (GE Healthcare, cat. no. 17‐5174‐01)
  • ClearSeal film (Hampton Research, cat. no. HR4‐521)
  • Ultracentrifuge Optima L‐80 (Beckman Instruments, cat. no. 355884)
  • FPLC (GE Amersham Biosciences, cat. no. 11001479 AKTAbasic UPC10 incl. Frac‐920)
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Literature Cited

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