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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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DSC analysis shows the biphasic thermal denaturation mode of the dcAvd/AVR4 protein.Heat-induced unfolding of the proteins was analysed by differential scanning calorimetry. The measured thermogram of dcAVD (A) and dcAVD/AVR4 (B) is shown without and with biotin (With Btn). Deconvoluted thermograms of dcAVD/AVR4 without (C) and with (D) biotin are also shown. The thermogram of dcAVD/AVR4 shows a melting point (Tm) at 91.3°C (C), which is about 11°C greater than for dcAVD (80.2°C, [11]). The smaller secondary peak shows a melting point at 86.3°C. Biphasic thermal denaturation mode is also detected in the presence of biotin (D); the melting point of the main peak is at 112.3°C, and the secondary peak is at 107.8°C. Interestingly, in the presence of biotin, dcAVD/AVR4 was denatured at a slightly lower temperature compared with dcAVD 115.9°C [11].
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pone-0020535-g005: DSC analysis shows the biphasic thermal denaturation mode of the dcAvd/AVR4 protein.Heat-induced unfolding of the proteins was analysed by differential scanning calorimetry. The measured thermogram of dcAVD (A) and dcAVD/AVR4 (B) is shown without and with biotin (With Btn). Deconvoluted thermograms of dcAVD/AVR4 without (C) and with (D) biotin are also shown. The thermogram of dcAVD/AVR4 shows a melting point (Tm) at 91.3°C (C), which is about 11°C greater than for dcAVD (80.2°C, [11]). The smaller secondary peak shows a melting point at 86.3°C. Biphasic thermal denaturation mode is also detected in the presence of biotin (D); the melting point of the main peak is at 112.3°C, and the secondary peak is at 107.8°C. Interestingly, in the presence of biotin, dcAVD/AVR4 was denatured at a slightly lower temperature compared with dcAVD 115.9°C [11].

Mentions: Differential scanning calorimetry (DSC) was used to analyse the thermodynamics of the heat-induced unfolding. In a DSC analysis of dcAVD/AVR4, a two-phase melting profile was observed both with and without biotin (Figure 5B). This melting profile was not detected in the dcAVD samples, in which the heat induced unfolding resulted in a single peak in the thermogram (Figure 5A). The thermograms recorded with dcAVD/AVR4 samples were deconvoluted in order to separate the two peaks (Figure 5C & Figure 5D). The main peak in the dcAVD/AVR4 thermogram revealed a melting point (Tm) at 91.3°C (Figure 5C), which is about 11°C higher compared to that measured for dcAVD (80.2°C). The smaller secondary peak showed a melting point at 86.3°C. In the presence of biotin, the main peak showed a melting point at 112.3°C, and a secondary peak at 107.8°C (Figure 5D). Interestingly, in the presence of biotin, dcAVD/AVR4 was denatured at a lower temperature compared to the melting temperature (115.9°C) of dcAVD. Overall, dcAVD/AVR4 had improved thermal stability in the absence of biotin, but the addition of biotin did not produce as significant thermal stabilization as in the case of dcAVD or wt AVD. Therefore, the exchange of the cp65-subunit of dcAVD with a circularly permuted AVR4 subunit increased the thermal stability of the chimeric fusion protein, while the other domain of the tandem gene remained unchanged (AVD-derived cp54). The lower biotin-binding affinity of the introduced AVR4-derived subunit reflected the thermal stability of the whole fusion protein in the presence of biotin. These results are clear indications of structural cooperativity between the subunits of the tetramer during thermal unfolding; however, previous studies have shown that (strept)avidin has relatively little or no structural cooperativity between subunits [21]–[23]. One reason for the apparently low cooperativity has been attributed to the subunit exchange between partially unfolded proteins [24]. For dcAVD, the covalent attachment of the subunits might block or at least significantly reduce the subunit exchange in the thermal unfolding process. Therefore, the dual chain concept allows a novel type of approach in studies elucidating the unfolding mechanisms of avidin proteins. The unfolding process was irreversible, which is typical for avidin proteins.


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)

DSC analysis shows the biphasic thermal denaturation mode of the dcAvd/AVR4 protein.Heat-induced unfolding of the proteins was analysed by differential scanning calorimetry. The measured thermogram of dcAVD (A) and dcAVD/AVR4 (B) is shown without and with biotin (With Btn). Deconvoluted thermograms of dcAVD/AVR4 without (C) and with (D) biotin are also shown. The thermogram of dcAVD/AVR4 shows a melting point (Tm) at 91.3°C (C), which is about 11°C greater than for dcAVD (80.2°C, [11]). The smaller secondary peak shows a melting point at 86.3°C. Biphasic thermal denaturation mode is also detected in the presence of biotin (D); the melting point of the main peak is at 112.3°C, and the secondary peak is at 107.8°C. Interestingly, in the presence of biotin, dcAVD/AVR4 was denatured at a slightly lower temperature compared with dcAVD 115.9°C [11].
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105096&req=5

pone-0020535-g005: DSC analysis shows the biphasic thermal denaturation mode of the dcAvd/AVR4 protein.Heat-induced unfolding of the proteins was analysed by differential scanning calorimetry. The measured thermogram of dcAVD (A) and dcAVD/AVR4 (B) is shown without and with biotin (With Btn). Deconvoluted thermograms of dcAVD/AVR4 without (C) and with (D) biotin are also shown. The thermogram of dcAVD/AVR4 shows a melting point (Tm) at 91.3°C (C), which is about 11°C greater than for dcAVD (80.2°C, [11]). The smaller secondary peak shows a melting point at 86.3°C. Biphasic thermal denaturation mode is also detected in the presence of biotin (D); the melting point of the main peak is at 112.3°C, and the secondary peak is at 107.8°C. Interestingly, in the presence of biotin, dcAVD/AVR4 was denatured at a slightly lower temperature compared with dcAVD 115.9°C [11].
Mentions: Differential scanning calorimetry (DSC) was used to analyse the thermodynamics of the heat-induced unfolding. In a DSC analysis of dcAVD/AVR4, a two-phase melting profile was observed both with and without biotin (Figure 5B). This melting profile was not detected in the dcAVD samples, in which the heat induced unfolding resulted in a single peak in the thermogram (Figure 5A). The thermograms recorded with dcAVD/AVR4 samples were deconvoluted in order to separate the two peaks (Figure 5C & Figure 5D). The main peak in the dcAVD/AVR4 thermogram revealed a melting point (Tm) at 91.3°C (Figure 5C), which is about 11°C higher compared to that measured for dcAVD (80.2°C). The smaller secondary peak showed a melting point at 86.3°C. In the presence of biotin, the main peak showed a melting point at 112.3°C, and a secondary peak at 107.8°C (Figure 5D). Interestingly, in the presence of biotin, dcAVD/AVR4 was denatured at a lower temperature compared to the melting temperature (115.9°C) of dcAVD. Overall, dcAVD/AVR4 had improved thermal stability in the absence of biotin, but the addition of biotin did not produce as significant thermal stabilization as in the case of dcAVD or wt AVD. Therefore, the exchange of the cp65-subunit of dcAVD with a circularly permuted AVR4 subunit increased the thermal stability of the chimeric fusion protein, while the other domain of the tandem gene remained unchanged (AVD-derived cp54). The lower biotin-binding affinity of the introduced AVR4-derived subunit reflected the thermal stability of the whole fusion protein in the presence of biotin. These results are clear indications of structural cooperativity between the subunits of the tetramer during thermal unfolding; however, previous studies have shown that (strept)avidin has relatively little or no structural cooperativity between subunits [21]–[23]. One reason for the apparently low cooperativity has been attributed to the subunit exchange between partially unfolded proteins [24]. For dcAVD, the covalent attachment of the subunits might block or at least significantly reduce the subunit exchange in the thermal unfolding process. Therefore, the dual chain concept allows a novel type of approach in studies elucidating the unfolding mechanisms of avidin proteins. The unfolding process was irreversible, which is typical for avidin proteins.

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