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Microbial Cell Factories Jan 2015Streptavidin is a tetrameric protein derived from Streptomyces avidinii, and has tight and specific biotin binding affinity. Applications of the streptavidin-biotin...
BACKGROUND
Streptavidin is a tetrameric protein derived from Streptomyces avidinii, and has tight and specific biotin binding affinity. Applications of the streptavidin-biotin system have been widely studied. Streptavidin is generally produced using protein expression in Escherichia coli. In the present study, the secretory production of streptavidin was carried out using Streptomyces lividans as a host.
RESULTS
In this study, we used the gene encoding native full-length streptavidin, whereas the core region is generally used for streptavidin production in E. coli. Tetrameric streptavidin composed of native full-length streptavidin monomers was successfully secreted in the culture supernatant of S. lividans transformants, and had specific biotin binding affinity as strong as streptavidin produced by E. coli. The amount of Sav using S. lividans was about 9 times higher than using E. coli. Surprisingly, streptavidin produced by S. lividans exhibited affinity to biotin after boiling, despite the fact that tetrameric streptavidin is known to lose its biotin binding ability after brief boiling.
CONCLUSION
We successfully produced a large amount of tetrameric streptavidin as a secretory-form protein with unique thermotolerance.
Topics: Amino Acid Sequence; Biotin; Electrophoresis, Polyacrylamide Gel; Molecular Sequence Data; Plasmids; Protein Binding; Protein Stability; Protein Structure, Quaternary; Recombinant Proteins; Streptavidin; Streptomyces lividans; Temperature
PubMed: 25582841
DOI: 10.1186/s12934-014-0188-y -
Molecules (Basel, Switzerland) Dec 2022Single molecule interactions between biotin and streptavidin were characterized with functionalized DeepTip probes and used as a model system to develop a comprehensive...
Single molecule interactions between biotin and streptavidin were characterized with functionalized DeepTip probes and used as a model system to develop a comprehensive methodology for the high-yield identification and analysis of single molecular events. The procedure comprises the covalent binding of the target molecule to a surface and of the sensing molecule to the DeepTip probe, so that the interaction between both chemical species can be characterized by obtaining force-displacement curves in an atomic force microscope. It is shown that molecular resolution is consistently attained with a percentage of successful events higher than 90% of the total number of recorded curves, and a very low level of unspecific interactions. The combination of both features is a clear indication of the robustness and versatility of the proposed methodology.
Topics: Microscopy, Atomic Force; Streptavidin; Biotin; Models, Biological
PubMed: 36615422
DOI: 10.3390/molecules28010226 -
ACS Nano May 2022Current strategies for the delivery of proteins into cells face general challenges of endosomal entrapment and concomitant degradation of protein cargo. Efficient...
Current strategies for the delivery of proteins into cells face general challenges of endosomal entrapment and concomitant degradation of protein cargo. Efficient delivery directly to the cytosol overcomes this obstacle: we report here the use of biotin-streptavidin tethering to provide a modular approach to the generation of nanovectors capable of a cytosolic delivery of biotinylated proteins. This strategy uses streptavidin to organize biotinylated protein and biotinylated oligo(glutamate) peptide into modular complexes that are then electrostatically self-assembled with a cationic guanidinium-functionalized polymer. The resulting polymer-protein nanocomposites demonstrate efficient cytosolic delivery of six biotinylated protein cargos of varying size, charge, and quaternary structure. Retention of protein function was established through efficient cell killing via delivery of the chemotherapeutic enzyme granzyme A. This platform represents a versatile and modular approach to intracellular delivery through the noncovalent tethering of multiple components into a single delivery vector.
Topics: Streptavidin; Biotin; Cytosol; Proteins; Polymers; Nanocomposites
PubMed: 35435664
DOI: 10.1021/acsnano.1c06768 -
Journal of Agricultural and Food... Apr 2022A simple and sensitive fluoroimmunoassay (FIA) based on a heavy-chain antibody (VHH) for rapid detection of fenitrothion was developed. A VHH library was constructed...
A simple and sensitive fluoroimmunoassay (FIA) based on a heavy-chain antibody (VHH) for rapid detection of fenitrothion was developed. A VHH library was constructed from an immunized alpaca, and one clone recognizing fenitrothion (namely, VHHjd8) was achieved after careful biopanning. It was biotinylated by fusing with the Avi tag and biotin ligase to obtain a fusion protein (VHHjd8-BT), showing both binding capacity to fenitrothion and the streptavidin poly-horseradish peroxidase conjugate (SA-polyHRP). Based on a competitive assay format, the absorbance spectrum of oxidized 3,3',5,5'-tetramethylbenzidine generated by SA-polyHRP overlapped the emission spectrum of carbon dots, which resulted in quenching of signals due to the inner-filter effect. The developed FIA showed an IC value of 1.4 ng/mL and a limit of detection of 0.03 ng/mL, which exhibited 15-fold improvement compared with conventional enzyme-linked immunosorbent assay. The recovery test of FIA was validated by standard GC-MS/MS, and the results showed good consistency, indicating that the assay is an ideal tool for rapid screening of fenitrothion in bulk food samples.
Topics: Enzyme-Linked Immunosorbent Assay; Fenitrothion; Fluoroimmunoassay; Single-Domain Antibodies; Streptavidin; Tandem Mass Spectrometry
PubMed: 35333506
DOI: 10.1021/acs.jafc.2c00826 -
Journal of the American Chemical Society Feb 2021Dinuclear iron centers with a bridging hydroxido or oxido ligand form active sites within a variety of metalloproteins. A key feature of these sites is the ability of...
Dinuclear iron centers with a bridging hydroxido or oxido ligand form active sites within a variety of metalloproteins. A key feature of these sites is the ability of the protein to control the structures around the Fe centers, which leads to entatic states that are essential for function. To simulate this controlled environment, artificial proteins have been engineered using biotin-streptavidin (Sav) technology in which Fe complexes from adjacent subunits can assemble to form [Fe-(μ-OH)-Fe] cores. The assembly process is promoted by the site-specific localization of the Fe complexes within a subunit through the designed mutation of a tyrosinate side chain to coordinate the Fe centers. An important outcome is that the Sav host can regulate the Fe···Fe separation, which is known to be important for function in natural metalloproteins. Spectroscopic and structural studies from X-ray diffraction methods revealed uncommonly long Fe···Fe separations that change by less than 0.3 Å upon the binding of additional bridging ligands. The structural constraints imposed by the protein host on the di-Fe cores are unique and create examples of active sites having entatic states within engineered artificial metalloproteins.
Topics: Biomimetic Materials; Biotin; Iron; Metalloproteins; Models, Molecular; Molecular Conformation; Streptavidin
PubMed: 33528256
DOI: 10.1021/jacs.0c12564 -
Scientific Reports Dec 2016The strong interaction between streptavidin (SA) and biotin is widely utilized in biotechnological applications. A SA variant, monovalent SA, was developed with a single...
The strong interaction between streptavidin (SA) and biotin is widely utilized in biotechnological applications. A SA variant, monovalent SA, was developed with a single and high affinity biotin-binding site within the intact tetramer. However, its structural characterization remains undetermined. Here, we seek to determine the crystal structure of monovalent SA at 1.7-Å resolution. We show that, in contrast to its 'close-state' in the only wild-type subunit, the L3,4 loops of three Dead SA subunits are free from crystal packing and remain in an 'open state', stabilized by a consistent H-bonding network involving S52. This H-bonding network also applies to the previously reported open state of the wild-type apo-SA. These results suggest that specific substitutions (N23A/S27D/S45A) at biotin-binding sites stabilize the open state of SA L3,4 loop, thereby further reducing biotin-binding affinity. The general features of the 'open state' SA among different SA variants may facilitate its rational design. The structural information of monovalent SA will be valuable for its applications across a wide range of biotechnological areas.
Topics: Binding Sites; Biotin; Crystallography, X-Ray; Models, Molecular; Protein Conformation; Streptavidin; Streptomyces
PubMed: 28000673
DOI: 10.1038/srep35915 -
Analytical Chemistry Apr 2011In recent years, there has been a growing interest in using porous microbeads such as agarose beads as solid supports to bind target molecules from complex fluid...
In recent years, there has been a growing interest in using porous microbeads such as agarose beads as solid supports to bind target molecules from complex fluid samples. Porous beads have large surface area to volume ratios and high receptor concentrations, and they facilitate relatively high sensitivity detection and multiplexing. Unfortunately, to take full advantage of the porous beads' attributes, long incubation times are needed due to the relatively slow mass transfer of target molecules from the exterior solution into the beads' interior. To accelerate the mass transfer process, we propose a novel assay in which functionalized porous beads are periodically compressed and expanded. Preliminary experiments were carried out to compare the performance of the pulsating beads with that of conventional, nonpulsating beads. These experiments indicate that the pulsating beads significantly accelerate binding rates with minimal increase in nonspecific binding. Thus, pulsing has the potential of significantly reducing assay time.
Topics: Biotin; Microfluidic Analytical Techniques; Particle Size; Porosity; Quantum Dots; Sepharose; Streptavidin; Surface Properties
PubMed: 21438559
DOI: 10.1021/ac200410v -
Clinical Chemistry and Laboratory... May 2017Immunoassays are now commonly used for hormone measurement, in high throughput analytical platforms. Immunoassays are generally robust to interference. However,... (Review)
Review
Immunoassays are now commonly used for hormone measurement, in high throughput analytical platforms. Immunoassays are generally robust to interference. However, endogenous analytical error may occur in some patients; this may be encountered in biotin supplementation or in the presence of anti-streptavidin antibody, in immunoassays involving streptavidin-biotin interaction. In these cases, the interference may induce both false positive and false negative results, and simulate a seemingly coherent hormonal profile. It is to be feared that this type of errors will be more frequently observed. This review underlines the importance of keeping close interactions between biologists and clinicians to be able to correlate the hormonal assay results with the clinical picture.
Topics: Biotin; False Negative Reactions; False Positive Reactions; Humans; Hyperthyroidism; Immunoassay; Streptavidin
PubMed: 27732554
DOI: 10.1515/cclm-2016-0606 -
Biomolecules Oct 2019Recent electronic transport experiments using metallic contacts attached to proteins identified some "stylized facts", which contradict conventional wisdom that...
Recent electronic transport experiments using metallic contacts attached to proteins identified some "stylized facts", which contradict conventional wisdom that increasing either the spatial distance between the electrodes or the temperature suppresses conductance exponentially. These include nearly temperature-independent conductance over the protein in the 30 to 300 K range, distance-independent conductance within a single protein in the 1 to 10 nm range and an anomalously large conductance in the 0.1 to 10 nS range. In this paper, we develop a generalization of the low temperature Landauer formula, which can account for the joint effects of tunneling and decoherence and can explain these new experimental findings. We use novel approximations, which greatly simplify the mathematical treatment and allow us to calculate the conductance in terms of a handful macroscopic parameters, instead of the myriads of microscopic parameters describing the details of an atomic level quantum chemical computation. The new approach makes it possible to get predictions for the outcomes of new experiments without relying solely on high performance computing and can distinguish important and unimportant details of the protein structures from the point of view of transport properties.
Topics: Cytochromes c; Electron Transport; Electrons; Myoglobin; Streptavidin
PubMed: 31614584
DOI: 10.3390/biom9100599 -
Biomolecules Apr 2022Intermittent jumping force is an operational atomic-force microscopy mode that produces simultaneous topography and tip-sample maximum-adhesion images based on force...
Intermittent jumping force is an operational atomic-force microscopy mode that produces simultaneous topography and tip-sample maximum-adhesion images based on force spectroscopy. In this work, the operation conditions have been implemented scanning in a repulsive regime and applying very low forces, thus avoiding unspecific tip-sample forces. Remarkably, adhesion images give only specific rupture events, becoming qualitative and quantitative molecular recognition maps obtained at reasonably fast rates, which is a great advantage compared to the force-volume modes. This procedure has been used to go further in discriminating between two similar protein molecules, avidin and streptavidin, in hybrid samples. The adhesion maps generated scanning with biotinylated probes showed features identified as avidin molecules, in the range of 40-80 pN; meanwhile, streptavidin molecules rendered 120-170 pN at the selected working conditions. The gathered results evidence that repulsive jumping force mode applying very small forces allows the identification of biomolecules through the specific rupture forces of the complexes and could serve to identify receptors on membranes or samples or be applied to design ultrasensitive detection technologies.
Topics: Avidin; Microscopy, Atomic Force; Streptavidin
PubMed: 35454182
DOI: 10.3390/biom12040594