Affimer proteins are versatile and renewable affinity reagents

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

doi:10.7554/elife.24903

Cited by 172 publications

(178 citation statements)

References 88 publications

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“…35 Given that Affimers express well in live cells, this approach should prove fruitful in identifying reagents that can be used to investigate cell biology that is dependent on related proteins, for instance signalling pathways where there is a need to discriminate the actions of highly related isoforms. 36 Comparison between the Affimer/BCL-xL and BCL-xL/BIM (PDB ID: 1PQ1) structures illuminates key features; the BH3 cleft narrows in response to Affimer binding in contrast to the wider cleft observed for binding of BIM (Fig. 5a).…”

Section: Discussion (Without Subheadings)mentioning

confidence: 94%

“…[47][48][49][50] Moreover, our own prior studies characterized BH3/BCL-2 family interactions as entropically driven. 20 Whilst the Affimer technology regularly produces binders with Kd in the nanomolar range 36 , here we added multiple layers of screening (inhibition of BH3 binding; compatible with anisotropy and ITC experiments) in addition to panning for high affinity binders. This will naturally lead to an attrition rate where clones that do not meet the criteria are lost, which may explain the slightly lower affinities we observe ( Table 1).…”

Section: Biophysical Analysis Of Affimersmentioning

confidence: 99%

“…[13][14][15][16] Multiple studies have endeavoured to identify chemotypes which mimic the BH3 domains so as to orthosterically inhibit BCL-2/BH3 PPIs including: constrained peptides, [17][18][19][20][21][22][23] peptidomimetics, [24][25][26][27] small molecules [28][29][30][31] and miniature proteins (identified with assistance from biological selection). 32,33 We have used a previously described Affimer library [34][35][36][37][38][39] to identify potent ligands for MCL-1, BCL-xL, BCL-2, BAK and BAX and selective inhibitors of MCL-1 and BCL-xL interactions with cognate BH3 partners.…”

Section: Introductionmentioning

confidence: 99%

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Selective Affimers Recognize BCL-2 Family Proteins Through Non-Canonical Structural Motifs

Miles¹,

Hóbor²,

Taylor³

et al. 2019

Preprint

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no more than 200 words)The BCL-2 family is a challenging set of proteins to target selectively due to sequence and structural homologies across the family. Selective ligands for the BCL-2 family regulators of apoptosis are desirable as probes to understand cell biology and apoptotic signalling pathways, and as starting points for inhibitor design. We have used phage display to isolate Affimer reagents (non-antibody binding proteins based on a conserved scaffold) to identify ligands for MCL-1, BCL-xL, BCL-2, BAK and BAX, then used multiple biophysical characterisation methods to probe the interactions. We established that purified Affimers elicit selective and potent recognition of their target BCL-2 protein. For anti-apoptotic targets, competitive inhibition of their canonical protein-protein interactions is demonstrated. Cocrystal structures reveal an unprecedented mode of molecular recognition; where a BH3 helix is normally bound, flexible loops from the Affimer dock into the BH3 binding cleft. Moreover, the Affimers induce a change in the target proteins towards a desirable drug bound like conformation. These results indicate Affimers can be used as alternative templates to inspire design of selective BCL-2 family modulators, and provide proof-ofconcept for the elaboration of selective non-antibody binding reagents for use in cell-biology applications. [198 words]

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Section: Discussion (Without Subheadings)mentioning

confidence: 94%

Section: Biophysical Analysis Of Affimersmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Selective Affimers Recognize BCL-2 Family Proteins Through Non-Canonical Structural Motifs

Miles¹,

Hóbor²,

Taylor³

et al. 2019

Preprint

Self Cite

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“…epifluorescence, ELISA or FACS), it is of major relevance for super-resolution microscopy techniques where the localization precision can be as high as 1 nm 7,8 . The linkage error can be reduced by using directly labeled small affinity probes like camelid single domain antibodies (sdAbs) also known as nanobodies (Nbs) 5,9 , affibodies 10 , aptamers 11,12 or affimers 13,14 , which all have sizes below 3 nm.…”

Section: Mainmentioning

confidence: 99%

Circumvention of common labeling artifacts using secondary nanobodies

Sograte‐Idrissi

Duque-Alfonso

Alevra

et al. 2019

Preprint

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The most common procedure to reveal the location of specific (sub)cellular elements in biological samples is via immunostaining followed by optical imaging. This is typically performed with target-specific primary antibodies (1.Abs), which are revealed by fluorophore-conjugated secondary antibodies (2.Abs). However, at high resolution this methodology can induce a series of artifacts due to the large size of antibodies, their bivalency, and their polyclonality. Here we use STED and DNA-PAINT super-resolution microscopy or light sheet microscopy on cleared tissue to show how monovalent secondary reagents based on camelid single-domain antibodies (nanobodies; 2.Nbs) attenuate these artifacts. We demonstrate that monovalent 2.Nbs have four additional advantages: 1) they increase localization accuracy with respect to 2.Abs; 2) they allow direct pre-mixing with 1.Abs before staining, reducing experimental time, and enabling the use of multiple 1.Abs from the same species; 3) they penetrate thick tissues efficiently; and 4) they avoid the artificial clustering seen with 2.Abs both in live and in poorly fixed samples. Altogether, this suggests that 2.Nbs are a valuable alternative to 2.Abs, especially when super-resolution imaging or staining of thick tissue samples are involved.

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“…Stefin A (see Table 1) [19,28]) have been generated against a number of Grb binding proteins [25] showing high selectivity of binding between isoforms that have approximately 70% sequence identity [55]. These Affimer reagents are specifically targeted to the Grb SH2 domains and are able to isolate their respective full length Grb proteins from U-2 OS cell lysates and are currently being tested for intracellular activity [25].…”

Section: For Example Affimer Proteins (Sbps Based On Plant Protein Pmentioning

confidence: 99%

Non-immunoglobulin scaffold proteins: Precision tools for studying protein-protein interactions in cancer

et al. 2018

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Cancer is frequently characterised by dysregulation of the cellular signalling processes that govern proliferation, survival and attachment. Understanding such dysregulation continues to present a challenge given the importance of protein-protein interactions in intracellular processes. Exploring this protein-protein interactome requires novel tools capable of discriminating between highly homologous proteins, individual domains and post-translational modifications. This review examines the potential of scaffold-based binding proteins to fulfil these requirements. It also explores protein-protein interactions in the context of intracellular signalling pathways and cancer, and demonstrates the uses of scaffold proteins as functional moderators, biosensors and imaging reagents. This review also highlights the timeliness and potential to develop international consortia to develop and validate highly specific "proteome" scaffold-based binding protein reagents with the ultimate aim of developing screening tools for studying the interactome.

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Affimer proteins are versatile and renewable affinity reagents

Cited by 172 publications

References 88 publications

Selective Affimers Recognize BCL-2 Family Proteins Through Non-Canonical Structural Motifs

Selective Affimers Recognize BCL-2 Family Proteins Through Non-Canonical Structural Motifs

Circumvention of common labeling artifacts using secondary nanobodies

Non-immunoglobulin scaffold proteins: Precision tools for studying protein-protein interactions in cancer

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