CaRA - A multi-purpose phage display library for selection of calcium-regulated affinity proteins.
N Biotechnol
; 72: 159-167, 2022 Dec 25.
Article
em En
| MEDLINE
| ID: mdl-36450334
ABSTRACT
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 subsequently be of great use in gentle protein purification or as novel therapeutic modalities.
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01-internacional
Base de dados:
MEDLINE
Assunto principal:
Bacteriófagos
/
Pró-Proteína Convertase 9
Idioma:
En
Revista:
N Biotechnol
Assunto da revista:
BIOLOGIA MOLECULAR
/
ENGENHARIA BIOMEDICA
Ano de publicação:
2022
Tipo de documento:
Article
País de afiliação:
Suécia