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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

Determination of ligand dissociation kinetics with a fluorescent-biotin conjugate.The dissociation of fluorescently labeled biotin from various avidin forms was studied by replacing the labeled biotin with an excess of free biotin. AVD, AVR4(C122S) and dcAVD showed a slow dissociation. For dcAVD/AVR4, a clearly biphasic dissociation process was observed. The analysis of the first part of the measurement (0–300 s, inset) reveals an estimate for the dissociation rate constant of 1.13×10−3 s−1 for the rapid dissociation phase of dcAVD/AVR4, which is about 100× greater when compared to avidin or AVR4(C122S).
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pone-0020535-g006: Determination of ligand dissociation kinetics with a fluorescent-biotin conjugate.The dissociation of fluorescently labeled biotin from various avidin forms was studied by replacing the labeled biotin with an excess of free biotin. AVD, AVR4(C122S) and dcAVD showed a slow dissociation. For dcAVD/AVR4, a clearly biphasic dissociation process was observed. The analysis of the first part of the measurement (0–300 s, inset) reveals an estimate for the dissociation rate constant of 1.13×10−3 s−1 for the rapid dissociation phase of dcAVD/AVR4, which is about 100× greater when compared to avidin or AVR4(C122S).

Mentions: Fluorescently labeled biotin and surface plasmon resonance (SPR) biosensor were used to study the biotin-binding characteristics of dcAVD/AVR4. In the experiment with fluorescently labeled biotin a bi-phasic ligand-dissociation process was detected, where roughly 50% of the protein subunits released biotin with rapid (kdiss = 1.1×10−3 s−1) dissociation kinetics (Figure 6). It is probable that the rapid biotin-dissociation phase was associated with the AVR4-derived cp65 domain. As we have previously shown, the biotin-binding affinity of the circularly permuted avidin cp65 is slightly less than wt AVD. In contrast, the cp54 version appears to behave more like wt AVD in terms of biotin-binding [9]. The previous studies have shown that the AVR4 protein has a slightly lower biotin-binding affinity (Kd = 3.6×10−14 M) than avidin (Kd = 1.1×10−16 M) [25]; however, the measured increase in the biotin dissociation rate of dcAVD/AVR4 was higher than expected based on previous studies, possibly reflecting the cooperativity between different parts of the biotin-binding site [26], [27].


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)

Determination of ligand dissociation kinetics with a fluorescent-biotin conjugate.The dissociation of fluorescently labeled biotin from various avidin forms was studied by replacing the labeled biotin with an excess of free biotin. AVD, AVR4(C122S) and dcAVD showed a slow dissociation. For dcAVD/AVR4, a clearly biphasic dissociation process was observed. The analysis of the first part of the measurement (0–300 s, inset) reveals an estimate for the dissociation rate constant of 1.13×10−3 s−1 for the rapid dissociation phase of dcAVD/AVR4, which is about 100× greater when compared to avidin or AVR4(C122S).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105096&req=5

pone-0020535-g006: Determination of ligand dissociation kinetics with a fluorescent-biotin conjugate.The dissociation of fluorescently labeled biotin from various avidin forms was studied by replacing the labeled biotin with an excess of free biotin. AVD, AVR4(C122S) and dcAVD showed a slow dissociation. For dcAVD/AVR4, a clearly biphasic dissociation process was observed. The analysis of the first part of the measurement (0–300 s, inset) reveals an estimate for the dissociation rate constant of 1.13×10−3 s−1 for the rapid dissociation phase of dcAVD/AVR4, which is about 100× greater when compared to avidin or AVR4(C122S).
Mentions: Fluorescently labeled biotin and surface plasmon resonance (SPR) biosensor were used to study the biotin-binding characteristics of dcAVD/AVR4. In the experiment with fluorescently labeled biotin a bi-phasic ligand-dissociation process was detected, where roughly 50% of the protein subunits released biotin with rapid (kdiss = 1.1×10−3 s−1) dissociation kinetics (Figure 6). It is probable that the rapid biotin-dissociation phase was associated with the AVR4-derived cp65 domain. As we have previously shown, the biotin-binding affinity of the circularly permuted avidin cp65 is slightly less than wt AVD. In contrast, the cp54 version appears to behave more like wt AVD in terms of biotin-binding [9]. The previous studies have shown that the AVR4 protein has a slightly lower biotin-binding affinity (Kd = 3.6×10−14 M) than avidin (Kd = 1.1×10−16 M) [25]; however, the measured increase in the biotin dissociation rate of dcAVD/AVR4 was higher than expected based on previous studies, possibly reflecting the cooperativity between different parts of the biotin-binding site [26], [27].

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