E. coli Expression and Purification of Fab Antibody Fragments

Ka Yin Kwong1, Christoph Rader1

1 Experimental Transplantation and Immunology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 6.10
DOI:  10.1002/0471140864.ps0610s55
Online Posting Date:  February, 2009
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Abstract

The Fab molecule was the first generated antibody fragment and still dominates basic research and clinical applications. This unit describes the E. coli expression and purification of Fab antibody fragments with and without a His tag, and is designed to yield sufficient protein for the evaluation and characterization of a panel of Fab selected from a Fab library by phage display. Curr. Protoc. Protein Sci. 55:6.10.1‐6.10.14. © 2009 by John Wiley & Sons, Inc.

Keywords: Fab; E. coli; His tag; IMAC; affinity chromatography

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: IMAC Purification of Fab‐(His)6 Proteins Following E. coli Expression
  • Alternate Protocol 1: Goat Anti–Human Fab Affinity Chromatography Following E. coli Expression
  • Support Protocol 1: Preparation of a Goat Anti–Human Fab Affinity Chromatography Column
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: IMAC Purification of Fab‐(His)6 Proteins Following E. coli Expression

  Materials
  • Glycerol stock of E. coli strain XL1‐Blue transformed with pC3C‐His encoding a Fab of interest (stored at −80°C; available from the authors through a Material Transfer Agreement)
  • LB agar plate with 100 µg/ml carbenicillin
  • SB medium (see recipe)
  • 100 µg/µl carbenicillin
  • 1 M isopropyl β‐D‐1‐thiogalactopyranoside (IPTG)
  • Phosphate‐buffered saline (PBS; appendix 2E)
  • HisTrap column washing buffer (see recipe)
  • HisTrap column elution buffer (see recipe)
  • 0.02% (w/v) sodium azide (optional)
  • 50‐ml conical tubes
  • 37°C shaking incubator
  • 2‐liter Erlenmeyer flasks
  • UV photometer (e.g., Eppendorf Biophotometer)
  • 50‐ to 2,000‐µl UVette disposable single‐sealed cuvettes (Eppendorf, cat. no. 95201005)
  • 500‐ml centrifuge bottles
  • Centrifuge equipped with a fixed‐angle rotor (e.g., Sorvall SLA‐3000 rotor)
  • Centrifuge equipped with a swing‐out rotor (e.g., Sorvall Legend/RT benchtop centrifuge)
  • 500‐ml, 0.45‐µm Stericup‐HV Filter Unit (Millipore, cat. no. SCHVU05RE)
  • 50‐ml, 0.45‐µm Steriflip Sterile Disposable Vacuum Filtration System (Millipore, cat. no. SE1M003M00)
  • 76‐mm Amicon Ultrafiltration Membranes with 10‐kDa MWCO (Millipore, cat. no. PBGC07610)
  • 400‐ml Amicon Stirred Cell 8400 (Millipore, cat. no. 5124)
  • Compressed nitrogen gas bottle with pressure regulator
  • Magnetic stirring plate
  • Peristaltic Pump P‐1 (GE Healthcare, cat. no. 18‐1110‐91) with 1.0‐mm (i.d.) silicone tubing (GE Healthcare, cat. no. 19‐4692‐01)
  • Peristaltic Pump P‐1 tubing connectors (GE Healthcare, cat. no. 19‐2150‐01)
  • 0.75‐mm (i.d.) 1/16” PEEK tubing (GE Healthcare, cat. no. 18‐1112‐53)
  • 1‐ml HisTrap FF crude column (GE Healthcare, cat. no. 11‐0004‐58) stored with 20% ethanol at 4°C
  • 15‐ml Amicon Ultra Centrifugal Filter Device with 10‐kDa MWCO (Millipore, cat. no. UFC901024)
  • Slide‐A‐Lyzer Dialysis Cassette (Extra Strength) with 10‐kDa MWCO and 3‐ to 12‐ml capacity (Pierce, cat. no. 66810), optional
  • 0.5‐ or 1.5‐ml microcentrifuge tubes
  • Additional reagents and equipment for running Coomassie‐stained SDS‐PAGE gels under reducing and nonreducing conditions (Chapter 10)

Alternate Protocol 1: Goat Anti–Human Fab Affinity Chromatography Following E. coli Expression

  • 0.5 M acetic acid
  • 1 M Tris⋅Cl, pH 8.0 ( appendix 2E)
  • NHS column storage buffer: 0.05 M Na 2HPO 4, 0.1% NaN 3, pH 7
  • 1‐ml HiTrap NHS‐activated HP column (GE Healthcare) coated with immobilized goat anti–human Fab polyclonal antibodies (see protocol 3)
  • 1.5‐ml microcentrifuge tubes

Support Protocol 1: Preparation of a Goat Anti–Human Fab Affinity Chromatography Column

  Materials
  • 1 mg/ml of either affinity‐purified goat anti–human Fab polyclonal antibodies (Bethyl Laboratories, cat. no. A80‐114A) or goat anti–human IgG, F(ab′) 2 fragment‐specific polyclonal antibodies (Jackson ImmunoResearch, cat. no. 109‐005‐006)
  • Column coupling buffer: 0.2 M NaHCO 3, 0.5 M NaCl (pH 8.3)
  • 1 mM ice‐cold HCl
  • NHS column buffer A: 0.5 M ethanolamine, 0.5 M NaCl (pH 8.3)
  • NHS column buffer B: 0.1 M acetate, 0.5 M NaCl (pH 4)
  • NHS column storage buffer: 0.05 M Na 2HPO 4, 0.1% NaN 3 (pH 7)
  • 15‐ml Amicon Ultra Centrifugal Filter Device with 30‐kDa MWCO membrane (Millipore, cat. no. UFC903024)
  • Centrifuge equipped with a swing‐out rotor (Sorvall Legend/RT benchtop centrifuge)
  • 1.5‐ml microcentrifuge tubes
  • Peristaltic Pump P‐1 (GE Healthcare, cat. no. 18‐1110‐91) with 1.0‐mm (i.d.) silicone tubing (GE Healthcare, cat. no. 19‐4692‐01)
  • Peristaltic Pump P‐1 tubing connectors (GE Healthcare, cat. no. 19‐2150‐01)
  • 1‐ml HiTrap NHS‐activated HP column (GE Healthcare, cat. no. 17‐0716‐01)
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Figures

Videos

Literature Cited

Literature Cited
   Hofer, T., Tangkeangsirisin, W., Kennedy, M.G., Mage, R.G., Raiker, S.J., Venkatesh, K., Lee, H., Giger, R.J., and Rader, C. 2007. Chimeric rabbit/human Fab and IgG specific for members of the Nogo‐66 receptor family selected for species cross‐reactivity with an improved phage display vector. J. Immunol. Methods 318:75‐87.
   Licht, J.J., Malecki, J.L., Agnew, H.D., Michelson‐Horowitz, D.J., and Tan, S. 2005. Comparison of affinity tags for protein purification. Protein Expr. Purif. 41:98‐105.
   Waugh, D.S. 2005. Making the most of affinity tags. Trends Biotechnol. 23:316‐320.
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