title: Construction of chimeric dual-chain avidin by tandem fusion of the related avidins.
abstract: BACKGROUND
  Avidin is a chicken egg-white protein with high affinity to vitamin H, also known as D-biotin. Many applications in life science research are based on this strong interaction. Avidin is a homotetrameric protein, which promotes its modification to symmetrical entities. Dual-chain avidin, a genetically engineered avidin form, has two circularly permuted chicken avidin monomers that are tandem-fused into one polypeptide chain. This form of avidin enables independent modification of the two domains, including the two biotin-binding pockets; however, decreased yields in protein production, compared to wt avidin, and complicated genetic manipulation of two highly similar DNA sequences in the tandem gene have limited the use of dual-chain avidin in biotechnological applications.
  
  
  RESULTS
  To overcome challenges associated with the original dual-chain avidin, we developed chimeric dual-chain avidin, which is a tandem fusion of avidin and avidin-related protein 4 (AVR4), another member of the chicken avidin gene family. We observed an increase in protein production and better thermal stability, compared with the original dual-chain avidin. Additionally, PCR amplification of the hybrid gene was more efficient, thus enabling more convenient and straightforward modification of the dual-chain avidin. When studied closer, the generated chimeric dual-chain avidin showed biphasic biotin dissociation.
  
  
  CONCLUSIONS
  The improved dual-chain avidin introduced here increases its potential for future applications. This molecule offers a valuable base for developing bi-functional avidin tools for bioseparation, carrier proteins, and nanoscale adapters. Additionally, this strategy could be helpful when generating hetero-oligomers from other oligomeric proteins with high structural similarity.
authors.affiliation: Institute of Biomedical Technology, University of Tampere and Tampere University Hospital, Tampere, Finland.
authors.forename: Tiina A
authors.initials: TA
authors.lastname: Riihimäki
authors.forename: Sampo
authors.initials: S
authors.lastname: Kukkurainen
authors.forename: Suvi
authors.initials: S
authors.lastname: Varjonen
authors.forename: Jarno
authors.initials: J
authors.lastname: Hörhä
authors.forename: Thomas K M
authors.initials: TK
authors.lastname: Nyholm
authors.forename: Markku S
authors.initials: MS
authors.lastname: Kulomaa
authors.forename: Vesa P
authors.initials: VP
authors.lastname: Hytönen
mesh_terms: D000818:Animals; D001360:Avidin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015374:Biosensing Techniques; D001710:Biotin / Q000235:genetics / Q000378:metabolism; D002645:Chickens; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D056004:Molecular Dynamics Simulation; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020349:Surface Plasmon Resonance
chemical_list: D011993:Recombinant Fusion Proteins; D001360:Avidin; D001710:Biotin