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Research Themes Protein design

Design and Evolution: Tunable Antibody Binders

SBKB [doi:10.1038/sbkb.2015.19]
Technical Highlight - June 2015
Short description: Reengineering the binding interface of Protein G yields a variant with stronger Fab binding and pH-tunable affinity.


Protein G (lavender) binds with low affinity to the constant domain of the antibody fragment (Fab; region CH1, green). Numbered residues (right) in the Protein-G interface were mutated to produce a tighter binding, pH-tunable form. Reprinted from 1 , with permission from Elsevier.

As the use of antibody reagents expand and evolve, the repertoire of antibody-binding proteins needs to keep pace. Streptococcal Protein G is a laboratory workhorse that targets IgG antibodies, binding both the fragment crystallizable (Fc) region with high affinity and the fragment antigen-binding (Fab) region with low affinity (Kd in micromolar range). To enable single-step purification of synthetic Fab antibodies generated using phage display, Kossiakoff and colleagues have developed a Protein-G reagent with superior Fab-binding properties.

The authors combined semirandom and rational approaches that focus on the 15 residues that lie within 5 Å of the interface of Protein G when bound to Fab. They screened a library of 3 billion variants of Protein G, with three rounds of 'bio-panning' enrichment for tight binding to Fab fragments using phage display—a method that links the sequence of a variant to its encoded protein during iterative selection steps.

A number of the interface residues contributed to a range of tighter binding, with the tightest binding variant, Protein G-A1, exhibiting 100-fold higher affinity (∼25 nM Kd). Further improvements eliminated Fc binding by targeted mutagenesis, and the resulting protein is thermostable and exhibits high solubility, promising to be suitable for purification, immunoprecipitation and as a chaperone for crystallization.

The team also noted that two residues in the Protein G-A1 interface with Fab are ionizable, suggesting a sensitivity to pH. They detected a 1,000-fold range of affinity from pH 7.5 to pH 4. In contrast, the affinity of wild-type Protein G exhibits broad robustness to pH. The dramatic pH dependence provides a switch that can be used for gentle protein elution during purification. This enabled the researchers to generate a convenient resin to purify Fab fragments in a single step, unlike Protein A–resin, which requires subsequent removal of degradation products from the antibody variable domain.

In the phage display experiment, two Asn residues known to cause high alkaline sensitivity were also mutated; introducing a change to Ala in Protein G-A1 conferred much greater alkaline stability, allowing for efficient cleanup of the Protein-G resin between runs. Finally, the authors joined affinity-matured Protein G-A1 dimers through a (Gly4Ser)3 tether, generating a ∼9-fold improvement in apparent affinity of the Fab–antigen interaction due to higher avidity. This robust and tunable binder will be a useful tool for antibody work.

Tal Nawy

References

  1. L.J. Bailey et al. Applications for an engineered Protein-G variant with a pH controllable affinity to antibody fragments.
    J Immunol Methods. 415, 24-30 (2014). doi:10.1016/j.jim.2014.10.003

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