Generating recombinant affinity reagents with arrayed targets
Methods for screening of affinity reagents for many target proteins of interest simultaneously. Arrayed targets (e.g., peptide, protein, RNA, cell, etc.) are used in affinity selection experiments to reduce the amount of target needed and to improve the throughput of discovering recombinant affinity reagents to a large collection of targets.
1. A method for affinity selection with arrayed target proteins, comprising the steps of:
a) providing an array comprising arrayed target proteins, wherein the arrayed target proteins comprise MAP2K5, CTBP1, SARA1A, CDK2 and RPS6KA3;
b) incubating a phage display material with the array;
c) washing the array to remove non-binding phage display material from the arrayed target proteins;
d) eluting from the array any binding phage display material;
e) amplifying the binding phage display material;
f) performing at least a further round of affinity selection repeating steps a)-d), whereby performing at least two rounds of affinity selection according to steps a) to d) yields bound phage display material having the highest affinity for at least one of the arrayed target proteins; and
g) isolating pools of the bound phage display material to identify phage display material having the highest affinity for the at least one of the arrayed target proteins.
2. The method of claim 1 , wherein providing an array of step (a) comprises:
i) transcribing and translating a nucleic acid molecule linked to a solid support, wherein the nucleic acid molecule encodes a fusion protein, the fusion protein comprising a target polypeptide and a first member of a protein binding pair;
wherein the first member of the protein binding pair binds to a second member of the protein binding pair, wherein the second member of the protein binding pair is linked to the solid support; wherein the arrayed target protein comprises the fusion protein bound to the solid support via the protein binding pair;
wherein the target polypeptides of the arrayed target proteins comprise MAP2K5, CTBP1, SRA1A, CDK2 and RPS6KA3.
3. The method of claim 1 , wherein identifying phage display material having the highest affinity for at least one of the arrayed target proteins comprises analyzing the isolated pools of bound phage display material via macrowell analysis or ELISA.
4. The method of claim 2 , wherein the first member of the protein binding pair comprises an antibody epitope, and the second member of the protein binding pair comprises an antibody.
5. The method of claim 4 , wherein the first member of the protein binding pair comprises a glutathione S-transferase (GST) epitope and the second member of the protein binding pair comprises an antibody that binds the GST epitope.
6. The method of claim 2 , wherein the first member of the protein binding pair comprises a Halo Tag ligand, and the second member of the protein binding pair comprises a Halo Tag.