A small bispecific protein selected for orthogonal affinity purification
Résumé
A novel protein domain with dual affinity has been created by randomization and selection. The small alkali stabilized albumin binding domain (ABD*) used as scaffold to construct the library, has affinity to human serum albumin (HSA) and is constituted of 46 amino acids of which 11 were randomized. To achieve a dual binder, the binding site of the inherent HSA-affinity was untouched and the randomization was made on the opposite side of the molecule. Despite its small size and randomization of almost 1/4th of its amino acids a bifunctional molecule, ABDz1, with ability to bind to both HSA and the Z2 domain/protein A was successfully selected using phage display. Moreover the newly selected variant showed improved affinity for HSA compared to the parental molecule. This novel protein domain has been characterized regarding secondary structure and affinity to the two different ligands. The possibility for orthogonal affinity purification has been investigated using two different affinity matrices representing the two ligands, the HSA-matrix and the protein A-based, MabSelect SuRe-matrix, and the new protein domain was purified to homogeneity. Furthermore, gene fusions between the new domain and three different target proteins with varying characteristics were made. Successful purification of all three versions could efficiently be carried out by an orthogonal affinity purification strategy taking advantage of the acquired affinities.
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