Affibodies – combinatorial binding proteins
The first non-immunoglobulin based framework for affinity proteins were described in
1995 (1). The new molecules were called affibodies and were based on combinatorial
protein engineering of the small and robust -helical structure of protein A (2).
Affibodies have since then found broad use as selective binding reagents in
biotechnology applications incl. separomics (6,8,11,12), diagnostics (10,13) and
receptor blocking (17). Recently, systems based on affibodies labelled for fluorescence
resonance energy transfer (FRET)-based detection have been developed to allow
quantitative measurements of non-labelled target molecules (9) for applications in for
example protein “chip” formats. So-called anti-idiotypic affibodies have also been
developed (7), suitable for Self-assembled Networks of Artificial Proteins (SNAP) to
allow the build-up of molecular micro structures (18). Recently, the structure of an
affibody-target co-complex was solved by both x-ray crystallography and NMR
(14,15). These structure showed upon a high charge and structural complementarity in
the binding interface, which had a size in comparity with antibody-antigen systems. In
parallel to the use of phage display technology, new selection systems for affibody
libraries have also been developed both based on Gram positive bacteria, FACSsorting of micro beads (16) and protein contact assays.
Key publications:
1. Nord, Nilsson, Nilsson, Uhlen & Nygren A combinatorial library of an alpha-helical bacterial receptor domain. Protein Eng 8,
601-608 (1995).
2. Nord et al. Binding proteins selected from combinatorial libraries of an alpha-helical bacterial receptor domain. Nature
Biotechnol 15, 772-777 (1997).
3. Nygren & Uhlen Scaffolds for engineering novel binding sites in proteins. Curr Opin Struct Biol 7, 463-469 (1997).
4. Hansson et al. An in vitro selected binding protein (affibody) shows conformation-dependent recognition of the respiratory
syncytial virus (RSV) G protein. Immunotechnology 4, 237-252 (1999).
5. Gunneriusson, Nord, Uhlen & Nygren Affinity maturation of a Taq DNA polymerase specific affibody by helix shuffling.
Protein Eng 12, 873-878 (1999).
6. Nord et al. Recombinant human factor VIII-specific affinity ligands selected from phage-displayed combinatorial libraries of
protein A. Eur J Biochem 268, 4269-4277 (2001).
7. Eklund, Axelsson, Uhlen & Nygren Anti-idiotypic protein domains selected from protein A-based affibody libraries. Proteins
48, 454-462 (2002).
8. Nord, K., Gunneriusson, E., Uhlén, M. and Nygren, P.-Å. (2000) "Ligands selected from combinatorial libraries of protein a for
use in affinity capture of apolipoprotein A-1 and Taq DNA polymerase" J. Biotechnol. 80, 45-54.
9. Karlström, A. and Nygren, P.-Å. [2001] "Dual labeling of a binding protein allows for specific fluorescence detection of native
protein" J. Anal. Biochem. 295, 22-30.
10. Rönnmark, J., Hansson, M., Nguyen, T., Uhlén, M., Robert, A., Ståhl, S. and Nygren, P.-Å. (2002) "Construction and
characterization of affibody-Fc chimeras produced in Escherichia coli" J. Immunol. Meth. 261, 199-211.
11. Rönnmark, J., Grönlund, H. Uhlén, M. and Nygren, P.-Å. (2002) "Human immunoglobulin A (IgA)-specific ligands from
combinatorial engineering of protein A" Eur. J. Biochem. 269, 2647-2655.
12. Gräslund, S., Eklund, M., Falk, R., Uhlén, M., Nygren, P.-Å. and Ståhl, S. (2002) "A novel affinity gene system allowing
protein A-based recovery of non-immunoglobulin gene products" J. Biotechnol. 99, 41.
13. Rönnmark, J, Kampf, C., Asplund, A., Höidén-Guthenberg, Wester, K., I-M, Pontén, F., Uhlén, M. and Nygren, P.-Å.
(2003)"Affibody--galactosidase immunoconjugates produced as soluble fusion proteins in the E. coli cytosol". J. Immunol. Meth.
281, 149-160.
14. Högbom, M., Eklund, M., Nygren, P.-Å. and Nordlund P. (2003) "Crystal structure of an in vitro evolved affibody-protein
complex" Proc. Natl. Acad. Sci. U.S.A. 100, 3191-3196.
15. Wahlberg, E., Lendel, C., Helgstrand, M., Allard, P., Dincbas-Renqvist, V., Hedqvist, A., Berglund, H., Nygren, P.-Å. and
Härd, T. (2003) "An affibody in complex with a target protein: structure and coupled folding" Proc. Natl. Acad. Sci. U.S.A. 100,
3185-3190.
16. Nord, N., Uhlén, M. and Nygren, P.-Å. (2003) "Microbead display of proteins by cell-free expression of anchored DNA" J.
Biotechnology 106, 1-13.
17. Sandström, K., Xu, Z., Forsberg, G. and Nygren, P.-Å. (2003) "Inhibition of the CD28-CD80 co-stimulation signal by a
CD28-binding ligand developed by combinatorial protein engineering". Protein Engineering 6, 691-697.
18. Eklund, M., Sandström K., Teeri, T. and Nygren, P.-Å. (2004) "Site-specific and reversible anchoring of active proteins onto
cellulose using a cellulosome-like complex" J. Biotechnol. In press.