Jump to content

Affimer

From Wikipedia, the free encyclopedia

This is an old revision of this page, as edited by Headbomb (talk | contribs) at 02:38, 5 February 2016 (→Structure and generation: clean up, replaced: Front Biosci. 1 (13) → Front Biosci. |volume=1 |issue=13 using AWB). The present address (URL) is a permanent link to this revision, which may differ significantly from the current revision.

The Affimer protein scaffold – showing the two loops and the amino terminus where designer or random peptides can be inserted to create the binding surface

An Affimer is a small, highly stable protein engineered to display peptide loops which provide a high affinity binding surface for a specific target protein. It is a protein of low molecular weight, 12–14 kDa,[1] derived from the cysteine protease inhibitor family of cystatins.[2][3][4][5]

Affimer proteins are composed of a scaffold, which is a stable protein based on the cystatin protein fold. They display two peptide loops and an N-terminal sequence that can be randomised to bind different target proteins with high affinity and specificity similar to antibodies. Stabilisation of the peptide upon the protein scaffold constrains the possible conformations which the peptide may take, thus increasing the binding affinity and specificity compared to libraries of free peptides.

Structure and generation

Affimer proteins were developed initially at the MRC Cancer Cell Unit in Cambridge then across two laboratories at the University of Leeds.[2][3][4][5] They are derived from cystatin proteins,[6] which function in nature as cysteine protease inhibitors,[7][8] and share the common tertiary structure of an alpha-helix lying on top of an anti-parallel beta-sheet.[9] These non-antibody scaffold proteins were engineered to be stable, non-toxic, biologically neutral and contain no post-translational modifications or disulphide bridges. Affimer technology makes use of two separate loop sequences, incorporating a total of 12 to 36 amino acids, to create a large potential target interaction surface of 650 to 1000 Å2, allowing for highly-specific, high affinity binding to target proteins.[2][5] Consequently, Affimer molecules can distinguish between proteins that differ by only a single amino acid, can detect subtle changes in protein expression levels even in a multiplexed format and can distinguish between multiple closely related protein domains. Phage display libraries of 1010 randomised potential target interaction sequences are generated and screened to identify the Affimer with high-specificity binding to the target protein and binding affinities in the nM range. The use of in vitro screening techniques allows affinity maturation to be performed to achieve even greater binding affinities and means that the target space is not limited by an animal host’s immune system. Affimer reagents have been produced to a large number of targets including ubiquitin chains,[10] immunoglobulins[11] and C-reactive protein,[12] for use in a number of molecular recognition applications.

Properties

Affimer proteins are recombinant. They display the robust characteristics of high thermostability, with a melting temperature over 80 °C,[13] resistance to extremes of pH,[13] freeze-thaw cycles and lyophilisation. The low molecular weight[14] of the Affimer means that problems of steric hindrance, typically observed with antibodies, are avoided across a number of binding applications. As an Affimer is generated using recombinant systems the generation is significantly more rapid and reproducible[15] compared with antibodies generated by immunisation of a host animal.

Applications

Affimer technology has been shown to function as research tools across a number of platforms, including ELISA,[16] Western blot,[17] surface plasmon resonance, affinity purification, Immunocytochemistry[18] and flow cytometry. Affimer reagents are readily isolated that inhibit protein-protein interactions[19] and can be expressed in mammalian cells to alter signalling pathways. In addition, they can be co-crystallised in complex with target proteins enabling drug discovery through in silico screening and displacement assays, making Affimer technology useful tools in drug target validation pipelines.

Affimer technology has been commercialised and developed by Avacta Life Sciences, who are developing them as reagents for research and diagnostic and as biotherapeutics.

References

  1. ^ Roberts, Josh P. (2013). "Biomarkers Take Center Stage". GEN. 33.
  2. ^ a b c Woodman R., Yeh J.T.-H., Laurenson S., Ko Ferrigno P. (2005). "Design and Validation of a Neutral Protein Scaffold for the Presentation of Peptide Aptamers". J Mol Biol. 352: 1118–1133. doi:10.1016/j.jmb.2005.08.001.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  3. ^ a b Hoffmann T., Stadler L.K.J., Busby M., Song Q., Buxton A.T., Wagner S.D., Davis J.J., Ko Ferrigno P. (2010). "Structure-function studies of an engineered scaffold protein derived from Stefin A. I: Development of the SQM variant". PEDS. 23 (5): 403–413.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  4. ^ a b Stadler L.K.J., Hoffmann T., Tomlinson D.C., Song Q., Lee T., Busby M., Nyathi Y., Gendra E., Tiede C., Flanagan K., Cockel S.J., Wipat A., Harwood C., Wagner S.D., Knowles M.A., Davis J.J., Keegan N., Ko Ferrigno P. (2011). "Structure-function studies of an engineered scaffold protein derived from Stefin A. II: Development and Applications of the SQT variant". PEDS. 24 (9): 751–763.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  5. ^ a b c Tiede C., Tang A.A., Deacon S.E., Mandal U., Nettleship J.E., Owen R.L., George S.E., Harrison D.J., Owens R.J., Tomlinson D.C., McPherson M.J. (2014). "Adhiron: A stable and versatile peptide display scaffold for molecular recognition applications". PEDS. 27 (5): 145–155.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  6. ^ "Affimers – Next Generation Affinity Reagents". Avacta Life Sciences. Retrieved 22 May 2014.
  7. ^ Turk V., Stoka V., Turk D. (2008). "Cystatins: Biochemical and structural properties, and medical releavnce". Front Biosci. 1 (13): 5406–5420.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  8. ^ Kondo H., Abe K., Emori Y., Arai S. (1991). "Gene organization of oryzastatin II, a new cystatin superfamily member of plant origin, is closely related to that of oryzacystatin-I but different from those of animal cystatins". FEBS Lett. 278: 87–90. doi:10.1016/0014-5793(91)80090-p.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  9. ^ Turk V. and W. Bode (1991). "The cystatins: protein inhibitors of cysteine proteinases". FEBS Lett. 285 (2): 213–219. doi:10.1016/0014-5793(91)80804-C. PMID 1855589.
  10. ^ Avacta Life Sciences. "Anti-diUbiquitin K48-linkage Affimer (36-28)".
  11. ^ Avacta Life Sciences. "Anti-Immunoglobulin Research Area of Affimers".
  12. ^ Johnson A, Song Q, Ko Ferrigno P, Bueno PR, Davis JJ. (Aug 7, 2012). "Sensitive Affimer and antibody based impedimetric label-free assays for C-reactive protein". Anal Chem. 84 (15): 6553–60. doi:10.1021/ac300835b. PMID 22789061.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  13. ^ a b Avacta Life Sciences. "Key benefits of Affimers - Robustness".
  14. ^ Avacta Life Sciences. "Key Benefits of Affimers - Small Size".
  15. ^ Avacta Life Sciences. "Key Benefits of Affimers - Rapid Generation".
  16. ^ Avacta Life Sciences. "ALS applications data - ELISA".
  17. ^ Avacta Life Sciences. "ALS applications data - Western blot".
  18. ^ Avacta Life Sciences. "ALS applications data - IHC".
  19. ^ Avacta Life Sciences. "ALS applications data - Protein-protein interactions".
Retrieved from "https://en.wikipedia.org/w/index.php?title=Affimer&oldid=703368554"