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Synthetic Antigen Binder (SAB) Generation and Crystallography

Project Description

The Synthetic Antigen Binder Generation and Crystallography core exploits technology established at the University of Chicago to generate designer binding proteins. The Koide and Kossiakoff groups, together with Sachdev S. Sidhu at the Univ of Toronto, have developed a simple but powerful strategy to design highly functional combinatorial libraries of antibodies and antibody-like binding proteins, collectively termed “synthetic antigen binders” or sABs. sABs are generated rapidly using phage-display technologies and in vitro selection, thus completely eliminating animal immunization, which is traditionally required for antibody generation. sABs are highly conformation-specific, and by fine-tuning the conditions under which they are generated, sABs can be engineered with specificity to the particular conformational state of a membrane protein of interest.

SABs have broad usage in biophysics, structural biology and cell biology. Our uses of sABs include (i) trapping a specific conformational state of membrane proteins for biophysical analyses and (ii) facilitating crystallization of membrane proteins as “crystallization chaperones.” Crystallization chaperones are a powerful tool because they can reduce conformational heterogeneity and increase protein crystallization. Crystallization chaperones mask counterproductive surfaces while extending surfaces predisposed to forming crystal contacts, and they also provide phasing information. The most relevant examples of the use of sABs as crystallization chaperones are the crystal structures for full-length KcsA in complex with an sAB.

The plan is to expose individual targets in each of the major functional classes to the selection process under conditions that stabilize a variety of conformations. sAB binders will then be identified for use in functional perturbation co-crystallization and eventual structural determinations.

References

  • Fellouse et al. High-throughput generation of synthetic antibodies from highly functional minimalist phage-displayed libraries. J Mol Biol. 2007;373:924-40.
  • Uysal et al. Crystal structure of full-length KcsA in its closed conformation. Proc Natl Acad Sci U S A. 2009;106:6644-9.
  • Koide. Engineering of recombinant crystallization chaperones. Curr Opin Struct Biol. 2009;19:449-57.