Search results for "Avidin"
showing 10 items of 97 documents
Targeting of biotinylated compounds to its target tissue using a low-density lipoprotein receptor–avidin fusion protein
2003
The very high binding affinity of avidin to biotin is one of the highest to occur in nature. We constructed a fusion protein composed of avidin and the endocytotic LDL receptor in order to target biotinylated molecules to cells of the desired tissues. In addition to the native avidin, charge-mutated and nonglycosylated avidins were utilized as part of the fusion proteins, in order to modify its properties. All of the fusion protein versions retained the biotin-binding capacity. Although the specificity was not increased, however, fusion proteins composed of natural avidin and nonglycosylated avidin bound most efficiently to the biotinylated ligands. Fluorescence microscopy and atomic force …
Efficient production of active chicken avidin using a bacterial signal peptide in Escherichia coli
2004
Chicken avidin is a highly popular tool with countless applications in the life sciences. In the present study, an efficient method for producing avidin protein in the periplasmic space of Escherichia coli in the active form is described. Avidin was produced by replacing the native signal sequence of the protein with a bacterial OmpA secretion signal. The yield after a single 2-iminobiotin–agarose affinity purification step was approx. 10 mg/l of virtually pure avidin. Purified avidin had 3.7 free biotin-binding sites per tetramer and showed the same biotin-binding affinity and thermal stability as egg-white avidin. Avidin crystallized under various conditions, which will enable X-ray cryst…
Real time optical immunosensing with flow-through porous alumina membranes
2014
Through the presentation of analytical data from bioassay experiments, measured by polarimetry, we demonstrate for the first time a real time immunoassay within a free standing macroporous alumina membrane. The 200 nm nominal pore diameter of the membrane enables flow-through, thereby providing an ideal fluidic platform for the targeted delivery of analytes to bioreceptors immobilized on the pore walls, enabling fast sensing response times and the use of small sample volumes (<100 mu L). For the immunoassay, the pore walls were first coated with the functional copolymer, copoly(DMA-NAS) using a novel coupling process, before immobilization of the allergen protein, beta-lactoglobulin, by spo…
Recent advances in surface plasmon resonance biosensors for microRNAs detection
2020
miRNAs are a large family of non-coding RNAs which play important roles in translational and post-transcriptional regulation of gene expression and biological processes. Abnormal expression of miRNAs is related to the initiation and progression of different diseases which make them be promising candidates for early medical diagnostics. Thus, accurate detection of miRNAs has great significance for disorder diagnosis. Nevertheless, their intrinsic characteristics such as short sequence, low concentration and sequence homology challenge routine techniques. The detection assays need to be extremely sensitive and selective in small value of intricate RNA samples. Biosensor-based strategies have …
High-resolution crystal structure of an avidin-related protein: insight into high-affinity biotin binding and protein stability.
2004
The chicken avidin gene belongs to an extended gene family encoding seven avidin-related genes (AVRs), of which only avidin is expressed in the chicken. The sequences of AVR4 and AVR5 are identical and the common protein (AVR4) has been expressed both in insect and bacterial systems. The recombinant proteins are similarly hyperthermostable and bind biotin with similarly high affinities. AVR4 was crystallized in the apo and biotin-complexed forms and their structures were determined at high resolution. Its tertiary and quaternary structures are very similar to those of avidin and streptavidin. Its biotin-binding site shows only a few alterations compared with those of avidin and streptavidin…
Specific interaction of desthiobiotin lipids and water-soluble biotin compounds with streptavidin
1991
As shown for biotin lipids (Ref. 1), the formation of perfect 2-D crystalline streptavidin domains can also be observed in the plane of desthiobiotin lipid monolayers. The binding constant of streptavidin with desthiobiotin (Ka = 5·1013 mol−1) is lower than that with biotin (Ka = 1015 mol−1) (Ref. 2). By adding free biotin into the subphase a competitive replacement and a detaching of the streptavidin domains from the desthiobiotin lipid monolayer takes place. Streptavidin domains built at receptor lipid monolayers are still functional. As could be shown, there are two biotin binding sites at each protein molecule that are fully accessible to biotin (Ref. 1). This can be proven by the inter…
Specific recognition and formation of two- dimensional streptavidin domains in monolayers: applications to molecular devices
1989
Abstract By virtue of the high-affinity specific interaction between the vitamin, biotin, and the protein, streptavidin, monolayers of synthetic lipids with biotin headgroups can tightly bind streptavidin at the lipid-water interface. Through this specific recognition fluorescently-labelled streptavidin spontaneously organizes in the plane of the interface to form large protein domains, directly visible in situ by fluorescence microscopy and exhibiting optical anisotropy. Further structural characterization has shown that these domains are two-dimensional protein crystals. Correlation with the known three-dimensional crystal structure of streptavidin indicates that two of streptavidin's fou…
Production of Hev b5 as a fluorescent biotin-binding tripartite fusion protein in insect cells
2005
The presented green fluorescent protein and streptavidin core-based tripartite fusion system provides a simple and efficient way for the production of proteins fused to it in insect cells. This fusion protein forms a unique tag, which serves as a multipurpose device enabling easy optimization of production, one-step purification via streptavidin-biotin interaction, and visualization of the fusion protein during downstream processing and in applications. In the present study, we demonstrate the successful production, purification, and detection of a natural rubber latex allergen Hev b5 with this system. We also describe the production of another NRL allergen with the system, Hev b1, which fo…
Mutation of a critical tryptophan to lysine in avidin or streptavidin may explain why sea urchin fibropellin adopts an avidin-like domain
1999
Sea urchin fibropellins are epidermal growth factor homologues that harbor a C-terminal domain, similar in sequence to hen egg-white avidin and bacterial streptavidin. The fibropellin sequence was used as a conceptual template for mutation of designated conserved tryptophan residues in the biotin-binding sites of the tetrameric proteins, avidin and streptavidin. Three different mutations of avidin, Trp-110-Lys, Trp-70-Arg and the double mutant, were expressed in a baculovirus-infected insect cell system. A mutant of streptavidin, Trp-120-Lys, was similarly expressed. The homologous tryptophan to lysine (W--K) mutations of avidin and streptavidin were both capable of binding biotin and bioti…
Pressure dependent arrangement of a protein in two-dimensional crystals specifically bound to a monolayer
1993
Abstract The arrangement of streptavidin bound to a biotinylated monolayer of a polymeric amphiphile at the air-water interface is studied as a function of lateral pressure or ligand density. Closely packed domains are observed by fluorescence microscopy. The arrangement of the protein in these domains is sensitively detected by X-ray reflectivity and an especially thorough data analysis yields the following: the distance of the protein from the air-monolayer interface varies with lateral pressure by 10 A; the interfaces involving the protein are much rougher than expected due to capillary waves; the electron density of the protein layer increases considerably on compression, which can be u…