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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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The performance of the chimeric dual-chain avidins in the PCR analysis and in E. coli expression.The usability of the generated chimeric dual chain avidin fusions was evaluated by amplifying the fusion genes by PCR and by expressing the proteins in E. coli. A) In the PCR analysis with primers recognizing regions inside the chimeric dcAVD genes (PCR 2), a clear difference between the behavior of the dcAVD genes (A) and the chimeric fusion genes (AA2, dcAVD/AVR2; AA4, dcAVD/AVR4; SA, dcAVD/SA) was detected. The chimeric fusion genes showed only two main PCR products; the appropriately sized product had the highest concentration. When the dcAVD gene was used as a template, several different-sized products were produced. The results from the PCR2 reaction (see Table S1) from two different conditions (I, II) are shown in the figure. (L, 1 kb DNA ladder). B) SDS-PAGE analysis of the purified chimeric dcAVDs showed that dcAVD/AVR4 was the most successfully expressed in its functional form in E. coli. The upper arrowhead indicates the location of the intact protein, and the lower arrowhead indicates the location of the proteolytically cleaved product. (M, molecular weight standard; AA2, dc-AVD-AVR2; AA4, dc-AVD-AVR4; AS, dc-AVD-SA, bA, chicken avidin control sample (protein expressed in E. coli [19]); cA, chicken avidin control sample).
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pone-0020535-g002: The performance of the chimeric dual-chain avidins in the PCR analysis and in E. coli expression.The usability of the generated chimeric dual chain avidin fusions was evaluated by amplifying the fusion genes by PCR and by expressing the proteins in E. coli. A) In the PCR analysis with primers recognizing regions inside the chimeric dcAVD genes (PCR 2), a clear difference between the behavior of the dcAVD genes (A) and the chimeric fusion genes (AA2, dcAVD/AVR2; AA4, dcAVD/AVR4; SA, dcAVD/SA) was detected. The chimeric fusion genes showed only two main PCR products; the appropriately sized product had the highest concentration. When the dcAVD gene was used as a template, several different-sized products were produced. The results from the PCR2 reaction (see Table S1) from two different conditions (I, II) are shown in the figure. (L, 1 kb DNA ladder). B) SDS-PAGE analysis of the purified chimeric dcAVDs showed that dcAVD/AVR4 was the most successfully expressed in its functional form in E. coli. The upper arrowhead indicates the location of the intact protein, and the lower arrowhead indicates the location of the proteolytically cleaved product. (M, molecular weight standard; AA2, dc-AVD-AVR2; AA4, dc-AVD-AVR4; AS, dc-AVD-SA, bA, chicken avidin control sample (protein expressed in E. coli [19]); cA, chicken avidin control sample).

Mentions: In contrast, when primers recognizing regions inside the tandem fusion gene were used, a clear difference in the behavior between dcAVD and the chimeric fusions was detected (Figure 2A). A number of side products were generated during the PCR amplification of the dcAVD gene, which was probably due to primers binding to multiple positions in the tandem gene and homologous recombination during the amplification process. In contrast, for the chimeric fusions, only two main PCR products were detected, which were the appropriately sized products. The dcAVD/SA showed the most efficient amplification. Using the chimeric fusion genes significantly improves PCR amplification and would allow targeting of mutagenesis to only a part of the gene, such as using the Quik-Change (Stratagene, La Jolla, CA, US) protocol. Moreover, this would enable the broader modification of dcAVD molecules, including targeted random mutagenesis of several amino acids.


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 performance of the chimeric dual-chain avidins in the PCR analysis and in E. coli expression.The usability of the generated chimeric dual chain avidin fusions was evaluated by amplifying the fusion genes by PCR and by expressing the proteins in E. coli. A) In the PCR analysis with primers recognizing regions inside the chimeric dcAVD genes (PCR 2), a clear difference between the behavior of the dcAVD genes (A) and the chimeric fusion genes (AA2, dcAVD/AVR2; AA4, dcAVD/AVR4; SA, dcAVD/SA) was detected. The chimeric fusion genes showed only two main PCR products; the appropriately sized product had the highest concentration. When the dcAVD gene was used as a template, several different-sized products were produced. The results from the PCR2 reaction (see Table S1) from two different conditions (I, II) are shown in the figure. (L, 1 kb DNA ladder). B) SDS-PAGE analysis of the purified chimeric dcAVDs showed that dcAVD/AVR4 was the most successfully expressed in its functional form in E. coli. The upper arrowhead indicates the location of the intact protein, and the lower arrowhead indicates the location of the proteolytically cleaved product. (M, molecular weight standard; AA2, dc-AVD-AVR2; AA4, dc-AVD-AVR4; AS, dc-AVD-SA, bA, chicken avidin control sample (protein expressed in E. coli [19]); cA, chicken avidin control sample).
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pone-0020535-g002: The performance of the chimeric dual-chain avidins in the PCR analysis and in E. coli expression.The usability of the generated chimeric dual chain avidin fusions was evaluated by amplifying the fusion genes by PCR and by expressing the proteins in E. coli. A) In the PCR analysis with primers recognizing regions inside the chimeric dcAVD genes (PCR 2), a clear difference between the behavior of the dcAVD genes (A) and the chimeric fusion genes (AA2, dcAVD/AVR2; AA4, dcAVD/AVR4; SA, dcAVD/SA) was detected. The chimeric fusion genes showed only two main PCR products; the appropriately sized product had the highest concentration. When the dcAVD gene was used as a template, several different-sized products were produced. The results from the PCR2 reaction (see Table S1) from two different conditions (I, II) are shown in the figure. (L, 1 kb DNA ladder). B) SDS-PAGE analysis of the purified chimeric dcAVDs showed that dcAVD/AVR4 was the most successfully expressed in its functional form in E. coli. The upper arrowhead indicates the location of the intact protein, and the lower arrowhead indicates the location of the proteolytically cleaved product. (M, molecular weight standard; AA2, dc-AVD-AVR2; AA4, dc-AVD-AVR4; AS, dc-AVD-SA, bA, chicken avidin control sample (protein expressed in E. coli [19]); cA, chicken avidin control sample).
Mentions: In contrast, when primers recognizing regions inside the tandem fusion gene were used, a clear difference in the behavior between dcAVD and the chimeric fusions was detected (Figure 2A). A number of side products were generated during the PCR amplification of the dcAVD gene, which was probably due to primers binding to multiple positions in the tandem gene and homologous recombination during the amplification process. In contrast, for the chimeric fusions, only two main PCR products were detected, which were the appropriately sized products. The dcAVD/SA showed the most efficient amplification. Using the chimeric fusion genes significantly improves PCR amplification and would allow targeting of mutagenesis to only a part of the gene, such as using the Quik-Change (Stratagene, La Jolla, CA, US) protocol. Moreover, this would enable the broader modification of dcAVD molecules, including targeted random mutagenesis of several amino acids.

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