Generation of High‐Specificity Antibodies against Membrane Proteins Using DNA‐Gold Micronanoplexes for Gene Gun Immunization

Debra T. Hansen1, Felicia M. Craciunescu1, Petra Fromme1, Stephen A. Johnston2, Kathryn F. Sykes3

1 Biodesign Center for Applied Structural Discovery, Arizona State University, Tempe, Arizona, 2 Biodesign Center for Innovations in Medicine, Arizona State University, Tempe, Arizona, 3 Current address: HealthTell, Inc., Chandler, Arizona
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 29.20
DOI:  10.1002/cpps.50
Online Posting Date:  February, 2018
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Membrane proteins are the molecular interface of the cell and its environs; however, studies of membrane proteins are highly technically challenging, mainly due to instability of the isolated protein. Towards the production of antibodies that recognize properly folded and stabilized forms of membrane protein antigen, we describe a DNA‐based immunization method for mice that expresses the antigen in the membranes of dendritic cells, thus allowing direct presentation to the immune system. This genetic immunization approach employs a highly efficient method of biolistic delivery based on DNA‐gold micronanoplexes, which are complexes of micron‐sized gold particles that allow dermal penetration and nanometer‐sized gold particles that provide a higher surface area for DNA binding than micron gold alone. In contrast to antibodies derived from immunizations with detergent‐solubilized protein or with protein fragments, antibodies from genetic immunization are expected to have a high capacity for binding conformational epitopes and for modulating membrane protein activity. © 2018 by John Wiley & Sons, Inc.

Keywords: antibodies; membrane protein; genetic immunization; DNA immunization; conformational epitopes; gene gun

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Preparation of DNA‐Gold Micronanoplex Bullets for Gene Gun Immunization
  • Basic Protocol 2: Gene Gun Immunization of Mice
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Preparation of DNA‐Gold Micronanoplex Bullets for Gene Gun Immunization

  • Ethanol, 200 proof
  • Acetone
  • Helium compressed gas, 99.99%, with helium pressure regulator (Bio‐Rad, cat. no. 165‐2413) that is specific for use with the Helios gene gun
  • Gold particles, unmodified, 1 to 2‐µm diameter (e.g., Bio‐Rad, cat. no. 165‐2264)
  • Concentrated sulfuric acid (98%)
  • 30% hydrogen peroxide, ACS grade
  • Water: use deionized or distilled water, except where molecular grade water is specified
  • 11‐mercaptoundecanoic acid, 95% (MUDA; e.g., Sigma‐Aldrich, cat. no450561; top off opened bottle with nitrogen gas, wrap lid with Parafilm, and store under desiccant; see Troubleshooting)
  • Nitrogen compressed gas, with nitrogen gas regulator
  • 0.1 M MES, pH 6.0 (see recipe)
  • N‐hydroxysuccinimide (NHS), 98% (e.g., Sigma, cat. no.130672; top off opened bottle with nitrogen gas, wrap lid with Parafilm, and store under desiccant; see Troubleshooting)
  • N‐(3‐dimethylaminopropyl)‐N′‐ethylcarbodiimide hydrochloride (EDC; e.g., Sigma, cat. no. E1769; top off opened bottle with nitrogen gas, wrap lid with Parafilm, store at −20°C under desiccant, and use within 6 months of purchase; see Troubleshooting)
  • 10% (w/v) polyethylenimine (PEI), pH 9 (see recipe)
  • Desiccant (e.g., Drierite 10‐20 mesh, indicating)
  • Dry ice (or −80°C freezer)
  • 508 mM HAuCl 4 (see recipe)
  • Cysteamine, ≥95% (CAS number 60‐23‐1; e.g., Sigma, cat. no. 30070, or Santa Cruz, cat. no. sc‐217991); store at 4°C under desiccant; use a recently‐ordered, unopened bottle
  • Sodium borohydride, 99.99% (NaBH 4; e.g., Sigma, cat. no. 480886; store under desiccant)
  • Purified plasmid DNAs (see Strategic Planning)
  • CpG oligodeoxynucleotides (see Strategic Planning and Troubleshooting)
  • 1‐butanol, ACS grade
  • Tefzel tubing, o.d. 0.125‐in., i.d. 0.093‐in., 50‐ft. per coil; Bio‐Rad, cat. no. 165‐2441 or K‐mac Plastics, cat. no. KM‐5374)
  • Vacuum line connected to two sequential vacuum trap flasks
  • Chemical fume hood
  • Flowmeter for helium gas, capable of 5 liters/min flowrate (e.g., Cole‐Parmer #EW‐68561‐57)
  • Analytical balance
  • Silicone (adapter) tubing: o.d. 5/32‐in., i.d. 3/32‐in., wall 1/32‐in., 50‐ft. per coil (e.g., Tygon 3350, Saint‐Gobain, cat. no.ABW00004); this tubing fits over the ends of the Tefzel tubing
  • 250‐ml glass Erlenmeyer flask
  • Paper towels
  • 50‐ml disposable conical polypropylene tubes suitable for centrifugation at 900 × g
  • Vortex mixer
  • Centrifuge for 50 ml polypropylene tubes, capable of 900 × g
  • Parafilm
  • Aluminum foil
  • Microtube mixer capable of 1400 rpm (e.g., Eppendorf Thermomixer)
  • Centrifuge for 15‐ml and 1.5‐ml polypropylene tubes, capable of 100 × g
  • Filters (0.22 µm) for sterilization of 0.1 M MES, pH 6.0 (e.g., Millipore bottle top filter, polyethersulfone, cat. no. SCGPT01RE)
  • Lyophilizer
  • −80°C freezer (or dry ice)
  • Kimwipes
  • Containers for storage of chemicals under desiccant at room temperature, at 4°C, and at −20°C
  • Micro magnetic stir bar, PTFE‐coated, 3‐mm diameter, 10‐mm length (e.g., Fisher, cat. no. 14‐513‐65SIX)
  • Magnetic stir plate
  • Single‐edged razor blades
  • 10‐ml syringes; non‐Luer Lok is recommended
  • 15‐ml and/or 1.5‐ml sterile disposable conical polypropylene centrifugation tubes (15‐ml tubes that have snap‐caps, e.g., Nunc, cat. no. 362694, will facilitate later steps during micronanoplex and bullet production)
  • Ethanol‐resistant marker (for labeling samples for steps during and after the preparation of DNA‐nanogold)
  • Forceps for handling bullets
  • Helios gene gun system Tubing Cutter (Bio‐Rad, cat. no. 165‐2422) with single‐edged razor blades; the Tubing Cutter is used to prepare the 1 cm length bullets
NOTE: Unless noted, all protocol steps are performed at room temperature.

Basic Protocol 2: Gene Gun Immunization of Mice

  • Six‐ to eight‐week‐old female A/J or BALB/c mice (e.g., from Jackson Laboratory) housed on a 12 hr dark/light schedule and provided food and water ad libitum; this age allows immunizations to be performed during the optimal age range of 8‐12 weeks (Hu, Yokoyama, & Kitagawa, ) following acclimatization and collection of pre‐immunization serum samples
  • Bullets (see protocol 1)
  • Forceps for handling bullets
  • Helios Gene Gun Kit (Bio‐Rad, cat. no. 165‐2411): includes gene gun, 5 O‐rings, 5 Barrel Liners, 5 Cartridge Holders, Cartridge Extractor Tool, and 9 V battery
  • Helium Hose Assembly with Swagelok Quick‐Connect fittings (Bio‐Rad, cat. no.165‐2412)
  • Helium Regulator (Bio‐Rad, cat. no. 165‐2413)
  • Hearing protection
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Key References
  Chambers & Johnston, 2003. See above.
  First description of the efficiency of genetic immunization towards the production of polyclonal antibodies against soluble proteins.
  Hansen et al., 2016. See above.
  First description of the efficiency of genetic immunization towards the production of polyclonal antibodies against membrane proteins.
  Liu et al., 2016. See above.
  Recent review of DNA‐based immunization methods specific for antibody production.
  Svarovsky et al., 2009. See above.
  First description of DNA‐gold micronanoplexes.
Internet Resources
  BioRad Helios gene gun manual.‐
  The PDF of the BioRad Helios gene gun manual.
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