The baculovirus expression vector system is a very powerful tool to produce virus‐like particles and gene‐therapy vectors, but the removal of coexpressed baculovirus has been a major barrier for wider industrial use. We used chimeric human immunodeficiency virus‐1 (HIV‐1) gag influenza‐hemagglutin virus‐like particles produced in Tnms42 insect cells using the baculovirus insect cell expression vector system as model virus‐like particles. A fast and simple purification method for these virus‐like particles with direct capture and purification within one chromatography step was developed. The insect cell culture supernatant was treated with endonuclease and filtered, before it was directly loaded onto a polymer‐grafted anion exchanger and eluted by a linear salt gradient. A 4.3 log clearance of baculovirus from virus‐like particles was achieved. The absence of the baculovirus capsid protein (vp39) in the product fraction was additionally shown by high performance liquid chromatography‐mass spectrometry. When considering a vaccination dose of 109 particles, 4200 doses can be purified per L pretreated supernatant, meeting the requirements for vaccines with <10 ng double‐stranded DNA per dose and 3.4 µg protein per dose in a single step. The process is simple with a very low number of handling steps and has the characteristics to become a platform for purification of these types of virus‐like particles. 相似文献
The downstream processing of enveloped virus‐like particles is very challenging because of the biophysical and structural similarity between correctly assembled particles and contaminating vesicular particles present in the feedstock. We used hydroxyl‐functionalized polymethacrylate monoliths, providing hydrophobic and electrostatic binding contributions, for the purification of HIV‐1 gag virus‐like particles. The clarified culture supernatant was conditioned with ammonium sulfate and after membrane filtration loaded onto a 1 mL monolith. The binding capacity was 2 × 1012/mL monolith and was only limited by the pressure drop. By applying either a linear or a step gradient elution, to decrease the ammonium sulfate concentration, the majority of double‐stranded DNA (88–90%) and host cell protein impurities (39–61%) could be removed while the particles could be separated into two fractions. Proteomic analysis and evaluation of the p24 concentration showed that one fraction contained majority of the HIV‐1 gag and the other fraction was less contaminated with proteins originated from intracellular compartments. We were able to process up to 92 bed volumes of conditioned loading material within 3 h and eluted in average 7.3 × 1011 particles per particle fraction, which is equivalent to 730 vaccination doses of 1 × 109 particles. 相似文献
Hepatitis B virus‐like particles expressed in Escherichia coli were purified using anion exchange adsorbents grafted with polymer poly(oligo(ethylene glycol) methacrylate) in flow‐through chromatography mode. The virus‐like particles were selectively excluded, while the relatively smaller sized host cell proteins were absorbed. The exclusion of virus‐like particles was governed by the accessibility of binding sites (the size of adsorbents and the charge of grafted dextran chains) as well as the architecture (branch‐chain length) of the grafted polymer. The branch‐chain length of grafted polymer was altered by changing the type of monomers used. The larger adsorbent (90 μm) had an approximately twofold increase in the flow‐through recovery, as compared to the smaller adsorbent (30 μm). Generally, polymer‐grafted adsorbents improved the exclusion of the virus‐like particles. Overall, the middle branch‐chain length polymer grafted on larger adsorbent showed optimal performance at 92% flow‐through recovery with a purification factor of 1.53. A comparative study between the adsorbent with dextran grafts and the polymer‐grafted adsorbent showed that a better exclusion of virus‐like particles was achieved with the absorbent grafted with inert polymer. The grafted polymer was also shown to reduce strong interaction between binding sites and virus‐like particles, which preserved the particles’ structure. 相似文献
Virus‐like particles have been successfully used as safe vaccines, as their structure is identical to their native counterparts but devoid of the viral genetic material. However, production of these complex structures is not easy, as recombinant proteins must assemble into virus‐like particles. Techniques to differentiate assembled and soluble proteins, as well as assembly intermediaries often present in a sample, are required. An example of complex virus‐like particles mixture occurs when rotavirus proteins are recombinantly expressed. Rotavirus‐like particles (RLP) can be single (sl), double (dl), or triple layered (tl). The use of RLP preparations as vaccines requires their complete characterization, including separation and quantification of each RLP in a sample. In this work, CZE was evaluated for the separation and quantification of dl and triple‐layered rotavirus‐like particles (tlRLP). A fused‐silica capillary with a deoxycholate running buffer efficiently separated dl and tlRLP in RLP preparations, as they migrated in two discrete peaks with electrophoretic mobilities of 1.24±0.04 and 2.95±0.03 Ti, respectively. Standard curves for dl and tlRLP were generated, and the response was linearly proportional to analyte concentration. The methodology developed was quantitative, specific, accurate, precise, and reproducible. CZE allowed the quantitative characterization of RLP preparations, which is required for evaluation of immunogens, for process development, and for quality control protocols. 相似文献
We discuss the purification of mono‐PEGylated HSA by hydrophobic interaction membrane chromatography. The hydrophobicity difference between the different fractionated species was induced by the addition of a lyotropic salt that caused phase transition of PEG (hydrophilic under normal condition) to a mildly hydrophobic form. The HSA PEGylation reaction mixture was mixed with lyotropic salt and passed through a stack of hydrophilized polyvinylidene fluoride membrane discs. Unmodified HSA was obtained in the flow through, while the PEGylated forms of the protein bound to the membrane and could be eluted by reducing the salt concentration. Among the three major PEGylated forms of HSA present in the feed (i.e. mono–, di–, and tri–), mono‐PEGylated HSA was eluted first and could be resolved from the others. The purified material was analyzed by SDS‐PAGE, dynamic light scattering, and SEC combined with multi‐angle light scattering. All these analytical techniques indicated the presence of species that has a molar mass consistent with mono‐PEGylated HSA. A scaled‐down version of the membrane chromatographic methods could be used for the rapid and sensitive analysis of PEGylated proteins. 相似文献
The growing demands of advanced fluorescence and super‐resolution microscopy benefit from the development of small and highly photostable fluorescent probes. Techniques developed to expand the genetic code permit the residue‐specific encoding of unnatural amino acids (UAAs) armed with novel clickable chemical handles into proteins in living cells. Here we present the design of new UAAs bearing strained alkene side chains that have improved biocompatibility and stability for the attachment of tetrazine‐functionalized organic dyes by the inverse‐electron‐demand Diels–Alder cycloaddition (SPIEDAC). Furthermore, we fine‐tuned the SPIEDAC click reaction to obtain an orthogonal variant for rapid protein labeling which we termed selectivity enhanced (se) SPIEDAC. seSPIEDAC and SPIEDAC were combined for the rapid labeling of live mammalian cells with two different fluorescent probes. We demonstrate the strength of our method by visualizing insulin receptors (IRs) and virus‐like particles (VLPs) with dual‐color super‐resolution microscopy. 相似文献
We developed a new method for real‐time, three‐dimensional tracking of fluorescent particles. The instrument is based on a laser‐scanning confocal microscope where the focus of the laser beam is scanned or orbited around the particle. Two confocal pinholes are used to simultaneously monitor regions immediately above and below the particle and a feedback loop is used to keep the orbit centered on the particle. For moderate count rates, this system can track particles with 15 nm spatial resolution in the lateral dimensions and 50 nm in the axial dimension at a temporal resolution of 32 ms. To investigate the interaction of the tracked particles with cellular components, we have combined our orbital tracking microscope with a dual‐color, wide‐field setup. Dual‐color fluorescence wide‐field images are recorded simultaneously in the same image plane as the particle being tracked. The functionality of the system was demonstrated by tracking fluorescent‐labeled artificial viruses in tubulin‐eGFP expressing HUH7 cells. The resulting trajectories can be used to investigate the microtubule network with super resolution.相似文献
A novel isolation strategy, online hyphenation of ultrasonic extraction, Sephadex LH‐20 column chromatography combined with high‐speed countercurrent chromatography, was developed for pure compounds extraction and purification. Andrographolide from Andrographis paniculata was achieved only in a single step purification protocol via the present strategy. The crude powder was ultrasonic extracted and extraction was pumped into Sephadex LH‐20 column directly to cut the nontarget fractions followed by the second‐dimensional high‐speed countercurrent chromatography, hyphenated by a six‐port valve equipped at the post‐end of Sephadex LH‐20 column, for the final purification. The results yielded andrographolide with the amount of 1.02 mg and a purity of 98.5% in a single step, indicating that the present method is effective to harvest target compound from medicinal plant. 相似文献
Precipitation polymerization is becoming increasingly popular in energy, environment and biomedicine. However, its proficient utilization highly relies on the mechanistic understanding of polymerization process. Now, a fluorescence self‐reporting method based on aggregation‐induced emission (AIE) is used to shed light on the mechanism of precipitation polymerization. The nucleation and growth processes during the copolymerization of a vinyl‐modified AIEgen, styrene, and maleic anhydride can be sensitively monitored in real time. The phase‐separation and dynamic hardening processes can be clearly discerned by tracking fluorescence changes. Moreover, polymeric fluorescent particles (PFPs) with uniform and tunable sizes can be obtained in a self‐stabilized manner. These PFPs exhibit biolabeling and photosensitizing abilities and are used as superior optical nanoagents for photo‐controllable immunotherapy, indicative of their great potential in biomedical applications. 相似文献
Porcine reproductive and respiratory syndrome (PRRS) virus is the causative agent of the most significant infectious disease currently affecting the swine industry worldwide. Density gradient ultracentrifugation remains the most commonly used method for porcine reproductive and respiratory syndrome virus (PRRSV) purification. However, this technique has notable drawbacks including long processing time and limited processing volume in each run. To overcome these limitations, a scalable process was developed. PRRSV propagated in MARC-145 was released by three freeze/thaw cycles. After a low speed centrifugation step, the virus particles in the supernatant were concentrated twice by an ultrafiltration step. The ultrafiltration step concentrated the virions effectively with no detectable loss while some cultural/cellular proteins were removed. The virions in the ultrafiltration retentate were then applied to a heparin affinity column on a fast performance liquid chromatography unit. The combined ultrafiltration and heparin affinity chromatography process removed more than 96% of cellular and medium proteins. During a stepwise elution strategy, the viral particles were eluted at two separate peaks recovering 27.5% and 25.4% of viral particles loaded onto the column with a purity of 194 and 3917 particles/μg protein, respectively. 相似文献
Due to the adsorption of biomolecules, the control of the biodistribution of nanoparticles is still one of the major challenges of nanomedicine. Poly(2‐ethyl‐2‐oxazoline) (PEtOx) for surface modification of nanoparticles is applied and both protein adsorption and cellular uptake of PEtOxylated nanoparticles versus nanoparticles coated with poly(ethylene glycol) (PEG) and non‐coated positively and negatively charged nanoparticles are compared. Therefore, fluorescent poly(organosiloxane) nanoparticles of 15 nm radius are synthesized, which are used as a scaffold for surface modification in a grafting onto approach. With multi‐angle dynamic light scattering, asymmetrical flow field‐flow fractionation, gel electrophoresis, and liquid chromatography‐mass spectrometry, it is demonstrated that protein adsorption on PEtOxylated nanoparticles is extremely low, similar as on PEGylated nanoparticles. Moreover, quantitative microscopy reveals that PEtOxylation significantly reduces the non‐specific cellular uptake, particularly by macrophage‐like cells. Collectively, studies demonstrate that PEtOx is a very effective alternative to PEG for stealth modification of the surface of nanoparticles.
In present study, a multiple columns and detectors liquid chromatography system for analysis of global components in traditional Chinese medicines was developed. The liquid chromatography system was consist of three columns, including size exclusion chromatography column, hydrophilic interaction chromatography column, and reversed phase chromato‐graphy column, and three detectors, such as diode array detector, evaporative light scattering detector, and mass spectrometry detector, based on column switching technique. The developed multiple columns and detectors liquid chromatography system was successfully applied to the analysis of global components, including macromolecular (polysaccharides), high (nucleosides and sugars)‐, and low (triterpenes)‐polarity small molecular compounds in Ganoderma, a well‐known Chinese medicinal mushroom. As a result, one macromolecular chromatographic peak was found in two Ganoderma species, 19 components were identified in Ganoderma lucidum (two sugars, three nucleosides, and 14 triterpenes), and four components (two sugars and two nucleosides) were identified in Ganoderma sinense. The developed multiple columns and detectors liquid chromatography system was helpful to understand comprehensive chemical characters in TCMs. 相似文献
A solid‐state fluorescent host system was created by self‐assembly of a 21‐helical columnar organic fluorophore composed of (1R,2S)‐2‐amino‐1,2‐diphenylethanol and fluorescent 1‐pyrenecarboxylic acid. This host system has a characteristic 21‐helical columnar hydrogen‐ and ionic‐bonded network. Channel‐like cavities are formed by self‐assembly of this column, and various guest molecules can be included by tuning the packing of this column. Moreover, the solid‐state fluorescence of this host system can change according to the included guest molecules. This occurs because of the change in the relative arrangement of the pyrene rings as they adjust to the tuning of the packing of the shared 21‐helical column, according to the size of the included guest molecules. Therefore, this host system can recognize slight differences in molecular size and shape. 相似文献
A series of methoxypolyethylene glycol‐terminated self‐fluorescent polyurethane multi‐block copolymers with excellent pH‐responsivity, self‐fluorescence, and biocompatibility are designed and synthesized. In our design, 1, 4‐bis (hydroxyethyl) piperazine is chosen as a pH‐responsive segment which can donate or accept protons in response to the change of environmental pH, and fluorescein isothiocyanate is used as a fluorescent dye conjugated into the micelles to offer self‐fluorescence. The chemical structure of the polyurethane multi‐block copolymers is characterized by nuclear magnetic resonance and Fourier transform infrared spectroscopy. The results of the acid‐based titration, the fluorescence spectrometry, and the ultraviolet visible spectroscopy indicate that the polyurethane multi‐block copolymers own an excellent pH‐buffering capacity responded to the change of pH values and the favorable self‐fluorescence property in an aqueous solution. And the ultraviolet absorption peaks of samples are strengthened with increasing of pH values, indicating that methoxypolyethylene glycol‐terminated self‐fluorescent polyurethane multi‐block copolymer can be a pH‐dependent fluorescent probe in a broad pH range. In addition, the in vitro cytotoxicity test showed that the polyurethane multi‐block copolymer has low cytotoxicity and good biocompatibility, which make it a promising nanoplatform for molecular imaging, diagnosis, and treatment of disease. 相似文献