title: Modification of the loops in the ligand-binding site turns avidin into a steroid-binding protein.
abstract: BACKGROUND
  Engineered proteins, with non-immunoglobulin scaffolds, have become an important alternative to antibodies in many biotechnical and therapeutic applications. When compared to antibodies, tailored proteins may provide advantageous properties such as a smaller size or a more stable structure.
  
  
  RESULTS
  Avidin is a widely used protein in biomedicine and biotechnology. To tailor the binding properties of avidin, we have designed a sequence-randomized avidin library with mutagenesis focused at the loop area of the binding site. Selection from the generated library led to the isolation of a steroid-binding avidin mutant (sbAvd-1) showing micromolar affinity towards testosterone (Kd ~ 9 μM). Furthermore, a gene library based on the sbAvd-1 gene was created by randomizing the loop area between β-strands 3 and 4. Phage display selection from this library led to the isolation of a steroid-binding protein with significantly decreased biotin binding affinity compared to sbAvd-1. Importantly, differential scanning calorimetry and analytical gel-filtration revealed that the high stability and the tetrameric structure were preserved in these engineered avidins.
  
  
  CONCLUSIONS
  The high stability and structural properties of avidin make it an attractive molecule for the engineering of novel receptors. This methodology may allow the use of avidin as a universal scaffold in the development of novel receptors for small molecules.
authors.affiliation: Institute of Biomedical Technology, University of Tampere and Tampere University Hospital, FI-33520 Tampere, Finland.
authors.forename: Tiina A
authors.initials: TA
authors.lastname: Riihimäki
authors.forename: Soili
authors.initials: S
authors.lastname: Hiltunen
authors.forename: Martina
authors.initials: M
authors.lastname: Rangl
authors.forename: Henri R
authors.initials: HR
authors.lastname: Nordlund
authors.forename: Juha A E
authors.initials: JA
authors.lastname: Määttä
authors.forename: Andreas
authors.initials: A
authors.lastname: Ebner
authors.forename: Peter
authors.initials: P
authors.lastname: Hinterdorfer
authors.forename: Markku S
authors.initials: MS
authors.lastname: Kulomaa
authors.forename: Kristiina
authors.initials: K
authors.lastname: Takkinen
authors.forename: Vesa P
authors.initials: VP
authors.lastname: Hytönen
mesh_terms: D001360:Avidin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D015723:Gene Library; D007700:Kinetics; D008024:Ligands; D019151:Peptide Library; D011485:Protein Binding; D015202:Protein Engineering; D020836:Protein Structure, Quaternary; D013739:Testosterone / Q000737:chemistry / Q000378:metabolism*
chemical_list: D008024:Ligands; D019151:Peptide Library; D001360:Avidin; D013739:Testosterone