13 research outputs found
Construction of Chimeric Dual-Chain Avidin by Tandem Fusion of the Related Avidins
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
Sequence features and evolutionary mechanisms in the chicken avidin gene family
The chicken avidin gene family comprises the avidin gene (avd) and
several homologous avidin-related genes (avrs). The sequences of the avr
genes are nearly identical to each other but exhibit nonrandomly
distributed, frequently nonsynonymous nucleotide substitutions compared
to avd. In this study, we determined the genetic distances and the
phylogeny of the avd and avr genes and found differences between
different exons and introns. Our results suggest the involvement of
biased gene conversion in the evolution of the genes. Furthermore, one
of the genes was identified as a putative fusion gene. The occurrence of
both gene conversion and recombination supports the models suggesting a
common initiation mechanism for conversion and crossing-over. The
existence of avidin-related proteins (AVRs) is currently unknown, but
the putative AVRs are expected to bind biotin similarly to avidin.
However, the observed sequence differences may affect the stability and
glycosylation patterns of the putative AVR proteins
The major conformational IgE-binding pitopes of hevein (Hev b6.02) are identified by a novel chimera-based allergen epitope mapping strategy
Isolated hevein like domains, but not 31-kd endochitinases, are responsible for IgE-mediated in vitro and invivo reactions in latex-fruit syndrome
Construction of hevein (Hev b 6.02) with reduced allergenicity for immunotherapy of latex allergy by comutation of six mino acid residues on the conformational IgE epitopes
Cloning and characterization of scavidin, a fusion protein for the targeted delivery of biotinylated molecules
We have constructed a novel fusion protein "Scavidin" consisting of the macrophage scavenger receptor class A and avidin. The Scavidin fusion protein is transported to plasma membranes where the avidin portion of the fusion protein binds biotin with high affinity and forms the basis for the targeted delivery of biotinylated molecules. Subcellular fractionation analysis, immuno-staining, and electron microscopy demonstrated endosomal localization of the fusion protein. According to pulse-labeling and cross-linking studies Scavidin is found as monomers (55 kDa), dimers, and multimers, of which the 220-kDa form was the most abundant. The biotin binding capacity and active endocytosis of the biotinylated ligands were demonstrated in rat malignant glioma. Local Scavidin gene transfer to target tissues could have general utility as a universal tool to deliver biotinylated molecules at systemic low concentrations for therapeutic and imaging purposes, whereby high local concentration is achieved
Purification and characterization of an adenotin-like adenosine binding protein from human platelets
A pathogen-induced gene of barley encodes a HSP90 homologue showing striking similarity to vertebrate forms resident in the endoplasmic reticulum
Structural characterization of core-bradavidin in complex with biotin
<div><p>Bradavidin is a tetrameric biotin-binding protein similar to chicken avidin and bacterial streptavidin, and was originally cloned from the nitrogen-fixing bacteria <i>Bradyrhizobium diazoefficiens</i>. We have previously reported the crystal structure of the full-length, wild-type (wt) bradavidin with 138 amino acids, where the C-terminal residues Gly129-Lys138 (“Brad-tag”) act as an intrinsic ligand (<i>i</i>.<i>e</i>. Gly129-Lys138 bind into the biotin-binding site of an adjacent subunit within the same tetramer) and has potential as an affinity tag for biotechnological purposes. Here, the X-ray structure of core-bradavidin lacking the C-terminal residues Gly114-Lys138, and hence missing the Brad-tag, was crystallized in complex with biotin at 1.60 Å resolution [PDB:4BBO]. We also report a homology model of rhodavidin, an avidin-like protein from <i>Rhodopseudomonas palustris</i>, and of an avidin-like protein from <i>Bradyrhizobium sp</i>. Ai1a-2, both of which have the Brad-tag sequence at their C-terminus. Moreover, core-bradavidin V1, an engineered variant of the original core-bradavidin, was also expressed at high levels in <i>E</i>. <i>coli</i>, as well as a double mutant (Cys39Ala and Cys69Ala) of core-bradavidin (CC mutant). Our data help us to further engineer the core-bradavidin–Brad-tag pair for biotechnological assays and chemical biology applications, and provide deeper insight into the biotin-binding mode of bradavidin.</p></div