Construction of Chimeric Dual-Chain Avidin by Tandem Fusion of the Related Avidins

Abstract

BackgroundAvidin 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 FindingsTo 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.SignificanceThe 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.

Artificial Avidin-Based Receptors for a Panel of Small Molecules

“…Avidin is a frequently utilized protein tool in a plethora of life science applications, including imaging, purification, labeling, targeting, and detection. , This chicken-derived tetrameric beta-barrel protein has many desirable properties: it is rather small in size (60 kDa), it can be efficiently produced in E. coli , its structure is well-known (e.g., [PDB: 1VYO]), and it is highly resistant to a wide range of denaturing conditions such as high temperatures, extreme pH values, and many organic solvents. , Furthermore, its structure–function relationship has been widely explored using rational mutagenesis, and several avidins with tailored and novel functions exist, such as dual-chain avidin, − single-chain avidin, extremely thermostable avidin, , and steroid-binding avidin, thus expanding even further the usability of this scaffold.…”

Molecular features of steroid-binding antidins and their use for assaying serum progesterone

“…The binding site of Avds has been engineered by both mutating single residues involved directly in biotin binding and by manipulating the loop regions of Avds important for ligand recognition and specificity [37,38]. The L3,4-loop in particular, with a vital role for biotin binding, has been an active target for mutagenesis [5,7–10]. In our recent study, we reported antidins, i .…”

Generic Method for Attaching Biomolecules via Avidin–Biotin Complexes Immobilized on Films of Regenerated and Nanofibrillar Cellulose

“…Recently, several modifications aiming to alter avidin–biotin binding properties have been introduced . For example, reversible systems with monomeric and whole avidins have been developed to allow for regeneration of the respective biointerface. − Bifunctional avidins with two different biotin binding pockets , and that bind covalently to biotin have been reported, and the thermal stability (denaturation temperature over 90°) of avidin has been improved via chemical modification …”