Affimer proteins are versatile and renewable affinity reagents.

Christian Tiede,Danah Alqallaf,Ruth E Hughes,Robert Bedford,Alexander Balls,Naomi Gibson,Matt Johnson,Nicola Ingram,Alistair Curd,Alastair Smith,Sandra Bell,Sreenivasan Ponnambalam,Paul Ko Ferrigno,Vikesh Patel,Imeshi Wijetunga,Michelle Peckham,Filomena Esteves,Sophie J Heseltine,Yashar Sadigh,Azhar Maqbool,Rebecca Ross,Louise Coletta ,Joanne E Nettleship ,Iain W Manfield ,Robin S Bon ,Michael A Harrison ,Laura C Zanetti-Domingues ,Margaret A Knowles ,Mark J Drinkhill ,Hannah F Kyle ,Jonathan D Lippiat ,Sarah A Goodchild ,G Rex Smith ,Sarah R Needham ,Raymond J Owens ,Geoffrey W Platt ,Thomas P Peacock ,Heather L Martin ,Marisa L Martin-Fernandez ,Anna A Tang ,Michael E Webb ,Michael J Mcpherson ,Raj Prasad ,Ashley P E Roberts ,Thomas J Taylor ,Darren C Tomlinson

doi:10.7554/elife.24903

Abstract

Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications.

Highlights

Our understanding of biological processes at the cellular level has been underpinned by the traditional disciplines of genetics, biochemistry, and molecular biology
We demonstrate the generation of Affimers against various target molecules, including a small organic molecule, and we report their use in a number of widely used biochemical and cell biology assays
We have addressed whether alternative binders can target a specific Src-Homology 2 (SH2) domain by selecting Affimers against a range of SH2 domains

Summary

Introduction

Our understanding of biological processes at the cellular level has been underpinned by the traditional disciplines of genetics, biochemistry, and molecular biology. Focus has shifted towards large-scale studies of genomes and transcriptomes, the latter as surrogates for cellular proteomes These combined with high-throughput protein interaction studies, have led to the new discipline of Systems Biology, where proteins are considered in the context of networks of biochemical and developmental pathways. In the network view of protein behaviour, each protein or protein isoform may participate in many protein-protein interactions but available tools that allow researchers to test hypotheses in the biological context are lacking Technologies such as RNAi and CRISPR-Cas that lower or ablate protein expression are important tools, but may cloud the interpretation of a proposed relationship between a given gene product or protein domain and the observed cellular phenotype. The generation of tools should have the ability to block proteinprotein interactions systematically without affecting expression levels

Methods

Results

Conclusion