Abstract:The study of basic cell ultrastructure and intracellular physiological functions has been greatly aided by detection and identification of single macromolecules. Since the current in situ labeling methods for directly correlative (light and electron) microscopy observations have a number of substantial limitations, the semiconductor nanocrystals quantum dots gain distinguished with long-term imaging and high photostability. The quantum dots (Qdots) have quickly filled in the role, being found to be superior to… Show more
“…9 were either round shaped and of about 250 nm in diameter (representatively marked by white arrows) or larger (400–800 nm, marked by green arrows) and irregular shaped. Complexes of primary and secondary antibody were reported to stretch out for up to 30 nm [ 65 ] which may result in a larger apparent diameter of antibody decorated individual VLP. Fig.…”
BackgroundChimeric virus-like particles (VLP) allow the display of foreign antigens on their surface and have proved valuable in the development of safe subunit vaccines or drug delivery. However, finding an inexpensive production system and a VLP scaffold that allows stable incorporation of diverse, large foreign antigens are major challenges in this field.ResultsIn this study, a versatile and cost-effective platform for chimeric VLP development was established. The membrane integral small surface protein (dS) of the duck hepatitis B virus was chosen as VLP scaffold and the industrially applied and safe yeast Hansenula polymorpha (syn. Pichia angusta, Ogataea polymorpha) as the heterologous expression host. Eight different, large molecular weight antigens of up to 412 amino acids derived from four animal-infecting viruses were genetically fused to the dS and recombinant production strains were isolated. In all cases, the fusion protein was well expressed and upon co-production with dS, chimeric VLP containing both proteins could be generated. Purification was accomplished by a downstream process adapted from the production of a recombinant hepatitis B VLP vaccine. Chimeric VLP were up to 95% pure on protein level and contained up to 33% fusion protein. Immunological data supported surface exposure of the foreign antigens on the native VLP. Approximately 40 mg of chimeric VLP per 100 g dry cell weight could be isolated. This is highly comparable to values reported for the optimized production of human hepatitis B VLP. Purified chimeric VLP were shown to be essentially stable for 6 months at 4 °C.ConclusionsThe dS-based VLP scaffold tolerates the incorporation of a variety of large molecular weight foreign protein sequences. It is applicable for the display of highly immunogenic antigens originating from a variety of pathogens. The yeast-based production system allows cost-effective production that is not limited to small-scale fundamental research. Thus, the dS-based VLP platform is highly efficient for antigen presentation and should be considered in the development of future vaccines.
“…9 were either round shaped and of about 250 nm in diameter (representatively marked by white arrows) or larger (400–800 nm, marked by green arrows) and irregular shaped. Complexes of primary and secondary antibody were reported to stretch out for up to 30 nm [ 65 ] which may result in a larger apparent diameter of antibody decorated individual VLP. Fig.…”
BackgroundChimeric virus-like particles (VLP) allow the display of foreign antigens on their surface and have proved valuable in the development of safe subunit vaccines or drug delivery. However, finding an inexpensive production system and a VLP scaffold that allows stable incorporation of diverse, large foreign antigens are major challenges in this field.ResultsIn this study, a versatile and cost-effective platform for chimeric VLP development was established. The membrane integral small surface protein (dS) of the duck hepatitis B virus was chosen as VLP scaffold and the industrially applied and safe yeast Hansenula polymorpha (syn. Pichia angusta, Ogataea polymorpha) as the heterologous expression host. Eight different, large molecular weight antigens of up to 412 amino acids derived from four animal-infecting viruses were genetically fused to the dS and recombinant production strains were isolated. In all cases, the fusion protein was well expressed and upon co-production with dS, chimeric VLP containing both proteins could be generated. Purification was accomplished by a downstream process adapted from the production of a recombinant hepatitis B VLP vaccine. Chimeric VLP were up to 95% pure on protein level and contained up to 33% fusion protein. Immunological data supported surface exposure of the foreign antigens on the native VLP. Approximately 40 mg of chimeric VLP per 100 g dry cell weight could be isolated. This is highly comparable to values reported for the optimized production of human hepatitis B VLP. Purified chimeric VLP were shown to be essentially stable for 6 months at 4 °C.ConclusionsThe dS-based VLP scaffold tolerates the incorporation of a variety of large molecular weight foreign protein sequences. It is applicable for the display of highly immunogenic antigens originating from a variety of pathogens. The yeast-based production system allows cost-effective production that is not limited to small-scale fundamental research. Thus, the dS-based VLP platform is highly efficient for antigen presentation and should be considered in the development of future vaccines.
“…The use of nanoparticles with different sizes is problematic for high-resolution multimodal imaging, for semiquantitative measurement of epitope numbers, or when epitope density is high . However, the emission color of bare CDs is closely related to their size, shape, and structures; i.e., the optical properties are dictated by the quantum confinement effect.…”
Highly luminescent ultrasmall carbon nanodots (CDs) have been prepared by one-step microwave-assisted pyrolysis and functionalized with fluorescein photosensitizer by a diazo-bond. The absorption edge of such prepared fluorescein− NN−CDs was red-shifted in comparison with the bare one. Nevertheless, the emission signal induced by the nanoparticle quantum-sized graphite structure was quenched due to photoisomerization of the diazo group at the photoexcited state. In order to restrict the photoisomerization, i.e., rotation around the nitrogen−nitrogen bond, the diazo group was fixed by a metal cation to form a complex compound or chelate. The obtained metal complex of fluorescein−NN−CDs shows an absorbance maximum the same as bare CDs but a recovered emission signal from the nanoparticle moiety, which was bathochromically shifted. They exhibit lower quantum yield in comparison with the bare CDs but better photostability toward emission quenching in nutrition cell culture. The formed photosensitizerconjugated nanoprobes were proposed as multifunctional fluorophores for intracellular in vivo imaging due to their attractive photophysical attributes and tunable and excitation-dependent emission. The bioapplication of photosensitizer-conjugated CDs was demonstrated as fluorescent tracers for endocytosis pathways in cultured Tobacco cells. Their successful staining and lower toxicity to the plant cells were compared with conventional quantum dots (CdSe/ZnS core−shell type, which caused an acute toxicological in vivo effect).
“…The development of the state‐of‐the‐art imaging techniques obviously is related on the availability of appropriate labels, such as nanoparticles (NPs) that combine small size and high labeling efficiency with ability to be distinguished by analytical electron microscope (Loukanov and Emin, ). The labeling efficiency of the nanoprobes depends on the NPs capability to interact with the target molecules placed in depth of the specimen, so that only exposed are easily labeled (Rotello, ).…”
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