Library-based methods and compositions for introducing molecular switch functionality into protein affinity reagents
Methods and compositions are disclosed for introducing molecular switch functionality into a protein affinity reagent to render its binding to a target molecule sensitive to an environmental trigger, such as pH, while maintaining binding affinity to the target molecule. Combinatorial libraries created by the method are also disclosed.
1. A method of selecting protein affinity reagents encoded by a synthetic combinatorial nucleic acid library, wherein the protein affinity reagents comprise a heavy chain variable domain antibody (VHH), wherein protein affinity reagents are selected that have molecular switch functionality sensitive to an environmental trigger, have a target binding interface, with a plurality of ionizable amino acid residues and wherein a specific target molecule is released from the protein affinity reagent in the presence of the environmental trigger, wherein the environmental trigger is pH or a metal ion, the method comprising:
(a) contacting the library with the target molecule to form binding complexes with protein affinity reagents, wherein the target binding interface comprises 30 to 35 amino acids;
(b) separating the binding complexes from unbound library protein affinity reagents; and
(c) eluting a population of the binding complexes that are sensitive to the environmental trigger.
2. The method of claim 1 , wherein the method further comprises step (d) selecting the population of eluted reagents on the basis of the ability to bind the target molecule in the presence of a specific environmental trigger and remain unbound in the absence of the environmental trigger.
3. The method of claim 1 , wherein the library is produced in a prokaryotic, eukaryotic or in vitro expression system, or by chemical synthesis.
4. The method of claim 1 , wherein the library is produced in a prokaryotic system.
5. The method of claim 1 , wherein the library is produced in a eukaryotic system.
6. The method of claim 1 , wherein the ionizable amino acid residues are selected from the group consisting of histidine, arginine, lysine, aspartic acid, and glutamic acid residues.
7. The method of claim 6 , wherein the ionizable amino acid residues are histidine residues.
8. The method of claim 6 , wherein the ionizable amino acid residues are arginine residues.
9. The method of claim 6 , wherein the ionizable amino acid residues are aspartic acid residues.
10. The method of claim 2 , wherein the method further comprises step (e) isolating the selected population of eluted reagents via purification.