Chapter 6
Isolation of Artificial Binding Proteins (Affimer Reagents)
for Use in Molecular and Cellular Biology
Anna A. S. Tang, Christian Tiede, Michael J. McPherson,
and Darren C. Tomlinson
Abstract
Artificial binding proteins have been validated as alternatives to antibodies in their use as research reagents
in molecular and cellular biology. For example, they have been used as inhibitors of protein–protein
interactions to modulate activity, to facilitate crystallization, and as probes for cellular imaging.
Phage display is a widely used approach for isolating target-specific binding reagents, and it has even been
used to isolate isoform-specific binding proteins and binders that can distinguish between highly homologous protein domains.
Here, we describe methods that have been employed in isolating highly specific artificial binding proteins
against a wide range of target proteins.
Key words Artificial binding protein, Affimer, Research reagents, Protein–protein interaction,
Inhibitor, Phage display, Panning, Phage ELISA
1 Introduction
Antibodies are the most widely used research reagents for protein
binding; however, they have numerous limitations and issues with
validation and reproducibility [1–3]. Consequently, artificial binding proteins such as DARPins [4], monobodies [5], and Affimer
reagents [6–9] have been developed as promising alternatives with
the added benefits of bacterial production for ease of manufacturability and mammalian cell expression to study protein function.
Affimer reagents are based on two types of closely related scaffolds—a mammalian scaffold based on Stefin A [6, 7] and the
Adhiron scaffold based on a consensus sequence of phytocystatins
[8]. Both scaffolds were engineered to constrain two variable peptide sequences for molecular recognition (Fig. 1) [8]. Affimer
reagents have been utilized as affinity reagents in various applications, for example, as detection reagents for diagnostics, as inhibitors of protein–protein interactions to modulate activity, as
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_6, © Springer Science+Business Media, LLC, part of Springer Nature 2021
105
Isolation of Artificial Binding Proteins (Affimer Reagents)
for Use in Molecular and Cellular Biology
Anna A. S. Tang, Christian Tiede, Michael J. McPherson,
and Darren C. Tomlinson
Abstract
Artificial binding proteins have been validated as alternatives to antibodies in their use as research reagents
in molecular and cellular biology. For example, they have been used as inhibitors of protein–protein
interactions to modulate activity, to facilitate crystallization, and as probes for cellular imaging.
Phage display is a widely used approach for isolating target-specific binding reagents, and it has even been
used to isolate isoform-specific binding proteins and binders that can distinguish between highly homologous protein domains.
Here, we describe methods that have been employed in isolating highly specific artificial binding proteins
against a wide range of target proteins.
Key words Artificial binding protein, Affimer, Research reagents, Protein–protein interaction,
Inhibitor, Phage display, Panning, Phage ELISA
1 Introduction
Antibodies are the most widely used research reagents for protein
binding; however, they have numerous limitations and issues with
validation and reproducibility [1–3]. Consequently, artificial binding proteins such as DARPins [4], monobodies [5], and Affimer
reagents [6–9] have been developed as promising alternatives with
the added benefits of bacterial production for ease of manufacturability and mammalian cell expression to study protein function.
Affimer reagents are based on two types of closely related scaffolds—a mammalian scaffold based on Stefin A [6, 7] and the
Adhiron scaffold based on a consensus sequence of phytocystatins
[8]. Both scaffolds were engineered to constrain two variable peptide sequences for molecular recognition (Fig. 1) [8]. Affimer
reagents have been utilized as affinity reagents in various applications, for example, as detection reagents for diagnostics, as inhibitors of protein–protein interactions to modulate activity, as
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_6, © Springer Science+Business Media, LLC, part of Springer Nature 2021
105