Tamavidin 2-REV: An engineered tamavidin with reversible biotin-binding capability

Takakura, Yoshimitsu; Sofuku, Kozue; Tsunashima, Masako

doi:10.1016/j.jbiotec.2013.01.006

Cited by 21 publications

(16 citation statements)

References 19 publications

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“…1,2 Numerous variants of streptavidin exist today as a result of the efforts to further extend its application by fine tuning its stability, biotin affinity, and oligomerization. [3][4][5][6][7][8] Recently, we engineered a 13 kDa structural monomer, mSA, by combining streptavidin and rhizavidin sequences, which is stable and binds biotin with low nanomolar affinity. 9 mSA was engineered because wt streptavidin contains multiple binding sites and can aggregate biotinylated ligands, which leads to unwanted side effects during certain applications.…”

Section: Introductionmentioning

confidence: 99%

“…For example, the noncovalent interaction between streptavidin and biotin, which is one of the most stable in nature, has been used extensively to design systems for detection and labeling . Numerous variants of streptavidin exist today as a result of the efforts to further extend its application by fine tuning its stability, biotin affinity, and oligomerization . Recently, we engineered a 13 kDa structural monomer, mSA, by combining streptavidin and rhizavidin sequences, which is stable and binds biotin with low nanomolar affinity .…”

Section: Introductionmentioning

confidence: 99%

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Structure‐based engineering of streptavidin monomer with a reduced biotin dissociation rate

et al. 2013

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We recently reported the engineering of monomeric streptavidin, mSA, corresponding to one subunit of wild type (wt) streptavidin tetramer. The monomer was designed by homology modeling, in which the streptavidin and rhizavidin sequences were combined to engineer a high affinity binding pocket containing residues from a single subunit only. Although mSA is stable and binds biotin with nanomolar affinity, its fast off rate (koff ) creates practical challenges during applications. We obtained a 1.9 Å crystal structure of mSA bound to biotin to understand their interaction in detail, and used the structure to introduce targeted mutations to improve its binding kinetics. To this end, we compared mSA to shwanavidin, which contains a hydrophobic lid containing F43 in the binding pocket and binds biotin tightly. However, the T48F mutation in mSA, which introduces a comparable hydrophobic lid, only resulted in a modest 20-40% improvement in the measured koff . On the other hand, introducing the S25H mutation near the bicyclic ring of bound biotin increased the dissociation half life (t½ ) from 11 to 83 min at 20°C. Molecular dynamics (MD) simulations suggest that H25 stabilizes the binding loop L3,4 by interacting with A47, and protects key intermolecular hydrogen bonds by limiting solvent entry into the binding pocket. Concurrent T48F or T48W mutation clashes with H25 and partially abrogates the beneficial effects of H25. Taken together, this study suggests that stabilization of the binding loop and solvation of the binding pocket are important determinants of the dissociation kinetics in mSA.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Structure‐based engineering of streptavidin monomer with a reduced biotin dissociation rate

et al. 2013

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“…[11][12][13][14][15][16][17][18] Given that the codon preferences among these mammals are similar, there is a good chance that mam-tam2A and mam-tam2B would also be highly expressed in the cells of these other mammals. One or two amino acids of mam-tam2A and mam-tam2B could be changed to create derivatives with a lower isoelectric point, 7) reversible biotin-binding capability, 8) or extremely high thermal stability.…”

Section: Discussionmentioning

confidence: 99%

“…Tamavidin 2 can be produced in abundance in its soluble form in Escherichia coli, and its crystallographic structure has been elucidated. 6) It has been further engineered by the use of site-directed mutagenesis to have even lower non-specific binding, 7) reversible biotin binding, 8) and extremely high thermal stability. 9) Tamavidin 2 may also be employed as an affinity tag for purification of proteins fused to it.…”

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High-level expression of tamavidin 2 in human cells by codon-usage optimization

Takakura

Katayama

Nagata

2015

Bioscience, Biotechnology, and Biochemistry

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Tamavidin 2 is a fungal protein that binds to biotin with an extremely high affinity. Tamavidin 2 is superior to avidin or streptavidin in terms of its low-level non-specific binding and high-level thermal stability. However, the gene for tamavidin 2 is highly expressed in Escherichia coli but not in mammalian cells, restricting its application as an affinity tag in mammalian cells. Here, we optimized the codon usage of tamavidin 2 for human cells and found that the resultant mutant expressed tamavidin 2 at approximately 30-fold higher level compared with the native gene. The protein thus produced in human cells could be purified by iminobiotin affinity chromatography, bound tightly to biotin, and was stable at high temperature (82 °C). This powerful technology for high-level expression of tamavidin 2 in mammalian cells will be of value in evaluating various fusion proteins produced in mammalian cells for numerous applications.

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“…Biotinylated peptides were captured using Tamavidin2-Rev. Tamavidin2-Rev can bind to biotin-labelled substances and can release them under high concentrations of free biotin (Takakura et al, 2013). The biotinylated peptides were eluted using 2 mM biotin, and the eluted peptides were analyzed using LC-MS/MS.…”

Section: Mass Spectrometry Analysis Of Airid-ikba-dependent Biotinylated Proteins In Cellsmentioning

confidence: 99%

Faculty Opinions recommendation of AirID, a novel proximity biotinylation enzyme, for analysis of protein-protein interactions.

Cole

2020

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature

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Proximity biotinylation based on Escherichia coli BirA enzymes such as BioID (BirA*)and TurboID is a key technology for identifying proteins that interact with a target protein in a cell or organism. However, there have been some improvements in the enzymes that are used for that purpose. Here, we demonstrate a novel BirA enzyme, AirID (ancestral BirA for proximity-dependent biotin identification), which was designed de novo using an ancestral enzyme reconstruction algorithm and metagenome data. AirID-fusion proteins such as AirID-p53 or AirID-IkBa indicated biotinylation of MDM2 or RelA, respectively, in vitro and in cells, respectively. AirID-CRBN showed the pomalidomide-dependent biotinylation of IKZF1 and SALL4 in vitro. AirID-CRBN biotinylated the endogenous CUL4 and RBX1 in the CRL4 CRBN complex based on the streptavidin pull-down assay. LC-MS/MS analysis of cells that were stably expressing AirID-IkBa showed top-level biotinylation of RelA proteins. These results indicate that AirID is a novel enzyme for analyzing protein-protein interactions.

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Tamavidin 2-REV: An engineered tamavidin with reversible biotin-binding capability

Cited by 21 publications

References 19 publications

Structure‐based engineering of streptavidin monomer with a reduced biotin dissociation rate

Structure‐based engineering of streptavidin monomer with a reduced biotin dissociation rate

High-level expression of tamavidin 2 in human cells by codon-usage optimization

Faculty Opinions recommendation of AirID, a novel proximity biotinylation enzyme, for analysis of protein-protein interactions.

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