Available online 28 November 2022

Combinatorial library of calcium-regulated affinity binders

Phage display selections for novel target specificities with metal-dependent binding

Highly stable binders with nanomolar calcium-dependent affinities to target of choice

Conditional binding for multiple purposes such as gentler protein purification

Switch-like properties for engineering of therapeutic protein domains

Protein activity regulated by interactions with metal ions can be utilized for many different purposes, including biological therapies and bioprocessing, among others. Calcium ions are known to interact with the frequently occurring EF-hand motif, which can alter protein activity upon binding through an induced conformational change. The calcium-binding loop of the EF-hand motif has previously been introduced into a small protein domain derived from staphylococcal Protein A in a successful effort to render antibody binding dependent on calcium. Presented here, is a combinatorial library for calcium-regulated affinity, CaRA, based on this domain. CaRA is the first alternative scaffold library designed to achieve novel target specificities with metal-dependent binding. From this library, several calcium-dependent binders could be isolated through phage display campaigns towards a set of unrelated target proteins (IgE Cε3-Cε4, TNFα, IL23, scFv, tPA, PCSK9 and HER3) useful for distinct applications. Overall, these monomeric CaRA variants showed high stability and target affinities within the nanomolar range. They displayed considerably higher melting temperatures in the presence of 1 mM calcium compared to without calcium. Further, all discovered binders proved to be calcium-dependent, with the great majority showing complete lack of target binding in the absence of calcium. As demonstrated, the CaRA library is highly capable of providing protein-binding domains with calcium-dependent behavior, independent of the type of target protein. These binding domains could be of great use subsequently in gentle protein purification or as novel therapeutic modalities.

ethylenediaminetetraacetic acid

size exclusion chromatography

surface plasmon resonance

variable temperature measurement

Protein engineering

calcium-dependent binding

Z-domain

phage display selection

© 2022 The Authors. Published by Elsevier B.V.