Table 3.

Outline of chaperone development using phage display for an experienced user.

Step	Considerations
Week 1. Generation of Chaperone Library
1. Select a chaperone scaffold format	Chaperone expression level & host, size, compatibility with different selection platforms, biophysical characteristics.
2. Clone the chaperone into a phagemid vector	Phagemid vector should be chosen based on desired monovalent or multivalent display
3. Generate a library of mutations based on the chaperone	Site-directed random mutagenesis of the binding site is an efficient method to engineer novel specificity and affinity for a chaperone. Library diversity can be limited so that the theoretical diversity can be efficiently sampled during panning.
4. Express & purify solubilized, native membrane protein for selection	Membrane protein can be locked in a particular state by complexation with agonist or antagonist to stabilize this state during chaperone selection and ultimately, crystallization.
Week 2–3. Clone Selection by Phage Panning
5. Transform phagemid library into E. coli	Maximize library size to be able to efficiently sample library diversity
6. Express chaperone-conjugated phage, incubate with membrane protein	The membrane protein can be immobilized in a plastic ELISA well for solid-phase selection or biotinylated for solution-phase selection followed by recovery using streptavidin-coated beads. Wash to remove non- and weakly-bound phage.
7. Elute bound phage, infect E. coli to repeat selection cycle	Repeat selection for additional rounds as necessary (often 3–5). Selection can be monitored by phage titer and/ or DNA sequencing. After several rounds, the recovered titer will typically increase and sequencing will reveal the presence of a few highly represented clones.
Week 4–6. Characterization of Chaperone Candidate
8. Screen selected clones from phage panning	Hundreds of clones can be efficiently screened using high-throughput methods, such as growth and binding assays in 96-well plates.
9. Express selected variants as soluble proteins	A few chaperone candidates are sequenced and sub-cloned for expression as a soluble chaperone.
10. Measure affinity and specificity for membrane protein	Candidate chaperones are characterized.
11. Optimize chaperone as necessary	Random mutagenesis of a lead chaperone can be used to improve both affinity and biophysical characteristics [77].
Week 7-?. Optimization and Screening of Co-crystallization Conditions