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Soili Lehtonen

University of Tampere
Supervisor: Vesa Hytönen, Markku S. Kulomaa
Funding: ISB
Date: 2010-06-29

Engineering novel avidin scaffold based binding proteins for small ligands

The homotetrameric chicken egg-white protein avidin binds vitamin B7, biotin, with extremely high affinity (Kd ~10^-15 M). Over the last few decades, avidin as well as its bacterial analogues (streptavidin, rhizavidin, bradavidin) and avidin related genes (AVRs) have been extensively characterized (Helppolainen et al. 2007, Keinänen et al. 1994, Laitinen et al. 2002, Nordlund et al. 2005b). Additionally, avidin and streptavidin have been used as powerful tools in different applications in medicine and biotechnology.

Avidin has a very stable structure making it an interesting target for protein engineering. The deep ligand binding pocket of avidin makes it optimal for binding small molecules. Broad knowledge about the ligand-binding characteristics of avidin as well as about interactions between avidin subunits have been obtained in various mutagenic studies. Avidin has been successfully modified over the years by targeted manipulations to adjust e.g. protein stability and binding properties and number of different kinds of useful mutants have been achieved (for review, see Laitinen et al. 2006). Further, developed dual-chain and single-chain avidin technology enables to construct pseudotetrameric structures combining subunits with different binding properties (Nordlund et al. 2004, Nordlund et al. 2005a).

The research project aims to develop novel and improved avidin scaffold based binders for different small ligands. Directed molecular evolution approaches like random mutagenesis and DNA shuffling (Stemmer 1994) will be used to construct DNA libraries employing AVRs and previously designed avidin mutants as source material. Phage display method (for review see Bratkovic 2010) will be used to screen the developed DNA libraries for binders which could be potentially used as tools in diagnostics or biotechnology.

References:

• Bratkovic, T. 2010. Progress in phage display: evolution of the technique and its application. Cell Mol.Life Sci. 67:749-767.
• Helppolainen, S.H., Nurminen, K.P., Maatta, J.A., Halling, K.K., Slotte, J.P., Huhtala, T., Liimatainen, T., Yla-Herttuala, S., Airenne, K.J., Narvanen, A., Janis, J., Vainiotalo, P., Valjakka, J., Kulomaa, M.S., Nordlund, H.R. 2007. Rhizavidin from Rhizobium etli: the first natural dimer in the avidin protein family. Biochem.J. 405: 397-405.
• Keinanen, R.A., Wallen, M.J., Kristo, P.A., Laukkanen, M.O., Toimela, T.A., Helenius, M.A., Kulomaa, M.S. 1994. Molecular cloning and nucleotide sequence of chicken avidin-related genes 1-5. Eur.J.Biochem. 220: 615-621.
• Laitinen, O.H., Hytonen, V.P., Ahlroth, M.K., Pentikainen, O.T., Gallagher, C., Nordlund, H.R., Ovod, V., Marttila, A.T., Porkka, E., Heino, S., Johnson, M.S., Airenne, K.J., Kulomaa, M.S. 2002. Chicken avidin-related proteins show altered biotin-binding and physico-chemical properties as compared with avidin. Biochem.J. 363: 609-617.
• Laitinen, O.H., Hytonen, V.P., Nordlund, H.R., Kulomaa, M.S. 2006. Genetically engineered avidins and streptavidins. Cell Mol.Life Sci. 63: 2992-3017.
• Nordlund, H.R., Hytonen, V.P., Horha, J., Maatta, J.A., White, D.J., Halling, K., Porkka, E.J., Slotte, J.P., Laitinen, O.H., Kulomaa, M.S. 2005a. Tetravalent single-chain avidin: from subunits to protein domains via circularly permuted avidins. Biochem.J. 392: 485-491.
• Nordlund, H.R., Hytonen, V.P., Laitinen, O.H., Kulomaa, M.S. 2005b. Novel avidin-like protein from a root nodule symbiotic bacterium, Bradyrhizobium japonicum. J.Biol.Chem. 280: 13250-13255.
• Nordlund, H.R., Laitinen, O.H., Hytonen, V.P., Uotila, S.T., Porkka, E., Kulomaa, M.S. 2004. Construction of a dual chain pseudotetrameric chicken avidin by combining two circularly permuted avidins. J.Biol.Chem. 279: 36715-36719.
• Stemmer, W.P. 1994. DNA shuffling by random fragmentation and reassemby: in vitro recombination for molecular evolution. Proc.Natl.Acad.Sci.U.S.A. 91: 10747-10751.