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

Riihimäki TA, Kukkurainen S, Varjonen S, Hörhä J, Nyholm TK, Kulomaa MS, Hytönen VP - PLoS ONE (2011)

Bottom Line: We observed an increase in protein production and better thermal stability, compared with the original dual-chain avidin.The improved dual-chain avidin introduced here increases its potential for future applications.Additionally, this strategy could be helpful when generating hetero-oligomers from other oligomeric proteins with high structural similarity.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedical Technology, University of Tampere and Tampere University Hospital, Tampere, Finland.

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.

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Related in: MedlinePlus

The homology model of dcAVD/AVR4 and the sequences of chimeric dcAVDs.The molecular model of dcAVD/AVR4 is generated by exploiting the existing 3-D structures of the dcAVD and AVR4. In the model, cpAVD is illustrated in light gray, and cpAVR4 is illustrated in dark gray. Amino acid sequence of cpAVD is in the light gray box, and the amino acid sequences of cpAVR2, cpAVR4, and cpSA are in the dark gray boxes. The linkers and the corresponding linker sequences connecting the original termini are shown in red. The linkers connecting the circularly permuted subunits and the corresponding linker sequences are shown in blue. The schematic representation of the protein expression cassette is in the bottom of the figure.
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pone-0020535-g001: The homology model of dcAVD/AVR4 and the sequences of chimeric dcAVDs.The molecular model of dcAVD/AVR4 is generated by exploiting the existing 3-D structures of the dcAVD and AVR4. In the model, cpAVD is illustrated in light gray, and cpAVR4 is illustrated in dark gray. Amino acid sequence of cpAVD is in the light gray box, and the amino acid sequences of cpAVR2, cpAVR4, and cpSA are in the dark gray boxes. The linkers and the corresponding linker sequences connecting the original termini are shown in red. The linkers connecting the circularly permuted subunits and the corresponding linker sequences are shown in blue. The schematic representation of the protein expression cassette is in the bottom of the figure.

Mentions: The chimeric dcAVD fusions were designed according to previously described principles [9]. The loop connecting the original termini of the circularly permuted SA, AVR4, and AVR2 was shortened when compared to the original dcAVD, as presented in Figure 1. This shortening was performed because the loop was largely invisible in the X-ray analysis of dcAVD, indicating high mobility of the loop region [10].


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

Riihimäki TA, Kukkurainen S, Varjonen S, Hörhä J, Nyholm TK, Kulomaa MS, Hytönen VP - PLoS ONE (2011)

The homology model of dcAVD/AVR4 and the sequences of chimeric dcAVDs.The molecular model of dcAVD/AVR4 is generated by exploiting the existing 3-D structures of the dcAVD and AVR4. In the model, cpAVD is illustrated in light gray, and cpAVR4 is illustrated in dark gray. Amino acid sequence of cpAVD is in the light gray box, and the amino acid sequences of cpAVR2, cpAVR4, and cpSA are in the dark gray boxes. The linkers and the corresponding linker sequences connecting the original termini are shown in red. The linkers connecting the circularly permuted subunits and the corresponding linker sequences are shown in blue. The schematic representation of the protein expression cassette is in the bottom of the figure.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105096&req=5

pone-0020535-g001: The homology model of dcAVD/AVR4 and the sequences of chimeric dcAVDs.The molecular model of dcAVD/AVR4 is generated by exploiting the existing 3-D structures of the dcAVD and AVR4. In the model, cpAVD is illustrated in light gray, and cpAVR4 is illustrated in dark gray. Amino acid sequence of cpAVD is in the light gray box, and the amino acid sequences of cpAVR2, cpAVR4, and cpSA are in the dark gray boxes. The linkers and the corresponding linker sequences connecting the original termini are shown in red. The linkers connecting the circularly permuted subunits and the corresponding linker sequences are shown in blue. The schematic representation of the protein expression cassette is in the bottom of the figure.
Mentions: The chimeric dcAVD fusions were designed according to previously described principles [9]. The loop connecting the original termini of the circularly permuted SA, AVR4, and AVR2 was shortened when compared to the original dcAVD, as presented in Figure 1. This shortening was performed because the loop was largely invisible in the X-ray analysis of dcAVD, indicating high mobility of the loop region [10].

Bottom Line: We observed an increase in protein production and better thermal stability, compared with the original dual-chain avidin.The improved dual-chain avidin introduced here increases its potential for future applications.Additionally, this strategy could be helpful when generating hetero-oligomers from other oligomeric proteins with high structural similarity.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedical Technology, University of Tampere and Tampere University Hospital, Tampere, Finland.

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.

Show MeSH
Related in: MedlinePlus