Construction of chimeric dual-chain avidin by tandem fusion of the related avidins

Tiina A. Riihimäki, Sampo Kukkurainen, Suvi Varjonen, Jarno Hörhä, Thomas K.M. Nyholm, Markku S. Kulomaa, Vesa P. Hytönen (Corresponding Author)

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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.

Principal Findings

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.

Significance

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.

Original languageEnglish
Article numbere20535
Number of pages10
JournalPLoS ONE
Volume6
Issue number5
DOIs
Publication statusPublished - 2011
MoE publication typeA1 Journal article-refereed

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Riihimäki, T. A., Kukkurainen, S., Varjonen, S., Hörhä, J., Nyholm, T. K. M., Kulomaa, M. S., & Hytönen, V. P. (2011). Construction of chimeric dual-chain avidin by tandem fusion of the related avidins. PLoS ONE, 6(5), [e20535]. https://doi.org/10.1371/journal.pone.0020535