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E. coli Expression and Purification of Fab Antibody Fragments

Ka Yin Kwong1,  Christoph Rader1

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

Basic Protocol: 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 -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: Goat Anti–Human Fab Affinity Chromatography Following E. coli Expression

 Additional Materials (also see Basic Protocol)
  • 0.5 M acetic acid
  • 1 M Tris×Cl, pH 8.0 (appendix 2E)
  • NHS column storage buffer: 0.05 M Na2HPO4, 0.1% NaN3, pH 7
  • 1-ml HiTrap NHS-activated HP column (GE Healthcare) coated with immobilized goat anti–human Fab polyclonal antibodies (see Support Protocol)
  • 1.5-ml microcentrifuge tubes

Support Protocol: 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 NaHCO3, 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 Na2HPO4, 0.1% NaN3 (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)
Looking for materials?  Suppliers Guide
     
 
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Figures

  • Figure 6.10.1
    Expression cassettes of pC3C (A) and pC3C-His (B). A single lac promoter (P lac) drives the synthesis of a dicistronic transcript. Two ribosome binding sites initiate the translation of two separate polypeptide chains, light chain VL-CL (white) and heavy chain fragment VH-CH1 (gray), which is fused to a (His)6-encoding sequence (H6) in pC3C-His. Through the leader peptides ompA and pelB, both polypeptides are transported to the periplasm of E. coli. A single disulfide bridge links CL and CH1 (not shown). For more information on pC3C in the context of phage display technology, see Hofer et al. (2007).

    View Image
  • Figure 6.10.2
    Flow chart of the protocols with time considerations. Gray areas indicate approximate time commitments on each of 5 days required for Fab expression and purification.

    View Image
  • Figure 6.10.3
    Analysis of Fab (left) and Fab-(His)6 (right) purified by goat anti–human Fab affinity chromatography (Alternate Protocol) and IMAC (Basic Protocol), respectively, by SDS-PAGE and Coomassie staining. Both proteins are derived from the same human anti-tetanus toxoid Fab that was selected from a naïve human Fab library by phage display. For each lane, 3 µg of protein in NuPAGE LDS Sample Buffer (Invitrogen, cat. no. NP007) with (red, reducing) or without (nonred, nonreducing) 50 mM DTT was separated by electrophoresis on a 1.5-mm, 10-well NuPage Novex 4% to 12% Bis Tris Gel (Invitrogen, cat. no. NP0335BOX) followed by staining with SimplyBlue SafeStain (Invitrogen, cat. no. LC6060). The molecular weight standard is given on the right in kDa. Note that reducing conditions split the ~50-kDa Fab protein into a ~25-kDa light chain and a ~25-kDa heavy chain fragment, which are linked by a single disulfide bridge under physiological conditions. The purity of Fab-(His)6 and Fab proteins purified by IMAC and goat anti–human Fab affinity chromatography, respectively, is comparable.

    View Image

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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