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Technology Topics Protein Expression

Caught in the Act

SBKB [doi:10.1038/sbkb.2012.175]
Technical Highlight - December 2013
Short description: Inventive selection strategies yield antibodies that capture distinctive states of target proteins.

sAB selection strategies. In competition selection, a competitor (red diamond) is added in excess to the solution along with an immobilized target (blue oval) and phage library (grey ovals). In subtractive selection, the phage library is added to an immobilized mimic, which effectively subtracts out all the binders to this competitor. Figure courtesy of Anthony Kossiakoff.

For most applications requiring monoclonal antibodies, synthetic antibodies (sABs), based on Fab antibody fragments generated using phage display, are more versatile and functional. Not only are such recombinant antibodies easy to produce in Escherichia coli; phage display inherently links phenotype and genotype so that sABs can be easily stored and reproduced. Importantly, the advantage of the selection process is that it can be fine-tuned to obtain sABs against desired functional states of a protein—from post-translational modifications (PTMs) to ligand-bound and conformation-specific or even complex-specific forms of the target. Now, Kossiakoff and colleagues (Chaperone-Enabled Studies of Epigenetic Regulation Enzymes) outline a strategy to do just that, starting from a synthetic Fab fragment library.

The authors designed two selection strategies to generate sABs that can discriminate between subtle differences in conformation or composition. For producing sABs that are region-specific or that can recognize surface properties, like PTMs or mutations, the target of interest is biotinylated and immobilized on magnetic beads. An excess amount of a soluble competitor, as the wild-type protein target in the case of PTMs and mutations, is added along with the phage display library. The competitor effectively “soaks up” the unwanted, nonspecific binders that do not recognize the desired epitope, and a pull-down of the target brings phages containing the target-specific sABs (left panel). To generate conformationally specific sABs, especially those that recognize conformations induced by ligand binding, an alternative selection can be used. For instance, to generate binders to the apo form of a protein, the competitor should be the ligand-bound form. However, if the ligand dissociates on a time scale that precludes isolation of binders to the apo form, the authors outline a two-step 'subtraction' strategy (right panel). First, a bead-bound competitor is used to “pre-clear” the phage pool; then, as in the one-step strategy above, the target is used to identify target-specific sABs. Following several iterations of subtraction and selection, identified binders to the desired form of the target are carefully validated.

The authors provide detailed step-by-step protocols and troubleshooting advice, in addition to discussing limitations of the system for selecting high-affinity binders for multi-protein complexes or for proteins with multiple post-translational modifications, among others. The approach can be adapted to yeast or mRNA display, and selections can be performed in high-throughput format.

Irene Kaganman

References

  1. M. Paduch et al. Generating conformation-specific synthetic antibodies to trap proteins in selected functional states.
    Methods. 60, 3-14 (2013). doi:10.1016/j.ymeth.2012.12.010

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