Computational design of ligand-binding proteins with high affinity and selectivity.

Engineered Digoxigenin binder protein DIG10.3

MNAKEIVVHALRLLENGDARGWSDLFHPEGVLEYPYPPPGYKTRFEGRETIWAHMRLFPEYMTIRFTDVQFYETADPDLA
IGEFHGDGVLTASGGKLAYDYIAVWRTRDGQILLYRLFFNPLRVLEPLGLEHHHHHH

Computationally designed based on hypothetical protein PA3332 from Pseudomonas aeruginosa

0.2M Ammonium acetate, 0.1M Bis-Tris pH5.5, 20% PEG3350, VAPOR DIFFUSION, HANGING DROP, temperature 298K

DIG10.3 WAS COMPUTATIONALLY ENGINEERED BASED ON PRE-DEFINED CRITERIA OF AFFINITY FOR DIGOXIGENIN. THE FOLLOWING MUTATIONS TO PA3332 (PDB ID 1Z1S) WERE FOUND TO MAXIMIZE BINDING AND OPTIMIZE PROTEIN STABILITY: L7V, S10A, C23S, F34Y, A37P, W41Y, H61Y, L62M, V64I, A90L, V92A, Q99Y, S103A, L105W, D117L, W119F, H124V, A127P, G130L AND V131E. TO AIDE IN CRYSTALLIZATION THE C-TERMINAL RESIDUES 132-141 WERE REMOVED AND REPLACED WITH 6X HIS-TAG

engineered, computationally designed, Digoxigenin-binding, DIGOXIGENIN BINDING PROTEIN

MNAKEILVHSLRLLENGDARGWCDLFHPEGVLEFPYAPPGWKTRFEGRETIWAHMRLFPEHLTVRFTDVQFYETADPDLA
IGEFHGDGVATVSGGKLAQDYISVLRTRDGQILLYRDFWNPLRHLEALGGV