Influence of DNA-protein interactions on purification and assembly of virus-like particles

Modular virus-like-particles (VLPs), presenting foreign antigens on their surface, are promising candidates for a wide range of future vaccines. A viable production pathway is the expression and purification of viral structural proteins and their subsequent in vitro assembly into VLPs, in a bioprocess environment. One promising approach is the use of murine polyomavirus major capsid protein VP1 as a carrier of modular epitopes from vaccine targets. This platform technology uses E. coli as an expression system and showed promising results in creating VLP vaccines candidates directed at influenza, Group A Streptococcus and other infectious pathogens. However, like other viral capsomeres and viral structures, purification using chromatography techniques remains a challenge as conventional high capacity ion exchange matrices suffer from low binding capacities. In this work the role of DNA-protein interaction during the purification of VLP precursor capsomeres is investigated. It is found that modular VP1 capsomeres coat DNA molecules forming large DNA-protein complexes that are unable to access the pores of chromatographic resins resulting in inefficient column binding. By increasing the salt concentration of the buffer above 0.3M NaCl, the DNA-protein complexes dissociate. At intermediate salt concentrations salt-tolerant ion-exchange resins can be used to efficiently capture and purify VP1 capsomeres, as the salt breaks the aggregates but is insufficient to interfere with binding to the salt-tolerant matrix. This approach increases the binding capacity of VLP precursor proteins by at least a magnitude over published laboratory-based methods. Please click Download on the upper right corner to see the full abstract

Changing Pattern of Characteristic Components in Black Garlic during Processing Analyzed by Ultra-high Performance Liquid Chromatography-Triple Quadrupole Tandem Mass Spectrometry

An analytical method based on ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-TQ-MS/MS) was established for the simultaneous determination of 10 flavor precursors and 21 free amino acids in black garlic. The method was applied for exploring the changing pattern of characteristic components in black garlic during processing. The results showed that the characteristic components in black garlic changed significantly during processing at 75 ℃ and 85% relative humidity. Among them, γ-aminobutyric acid, S-allyl-L-cysteine, isoalliin, glutamine, methiin, alliin, tryptophan, and γ-L-glutamyl-S-allyl-L-cysteine changed most obviously, and were identified as the chemical markers of changes in small molecular metabolites during the processing of black garlic. The established method has high sensitivity and accuracy, and can meet the detection requirements

Partial purification of alpha-amylase from culture supernatant of Bacillus subtilis in aqueous two-phase systems

The original publication can be found at www.springerlink.comA study was made of the partition and purification of -amylase from a culture supernatant of Bacillus subtilis in the polyethylene glycol (PEG)—citrate aqueous two-phase system (ATPS). Factors that influenced the partition of the protein in this system, including the molecular weight of the PEG, the tie line length of ATPS, the pH value and the sodium chloride concentration, were investigated. Purification of -amylase was attained with a purification factor (PF) of 1.8 and 90% yield at pH 6.0 in a PEG1000-citrate ATPS with short tie line length. By utilizing the salt-out effect of neutral salt, the purification of -amylase was further improved to 2.0 of PF and 80% yield in a PEG3350-citrate ATPS with 4% sodium chloride.Wenbo Zhi, Jiangnan Song, Jingxiu Bi and Fan Ouyan

New bio-based polymeric thermoset synthesized by ring-opening polymerization of soybean oil-based resin with green curing agent

Renewable resources, such as vegetable oils, woody biomass and terpenes, have attracted a great deal of attention in polymer synthesis [1, 2]. As one of the most important derivatives from rosin, the maleopimaric acid (MPA) is a fascinating choice to be a green curing agent for epoxy. In our study, MPA was successfully synthesized between abietic acid (AA) and maleic anhydride (MA) by using the catalyst p-toluene sulfonic acid[3]. The optimal experiment was conducted at 1:1.1 molar ratio of AA to MA and 190℃ in an airtight reactor for 2h. The chemical structure and properties of the product were characterized in detail by FT-IR, DSC, 1H NMR and 13C NMR, which indicated that MPA with high purity could be synthesized through the isomerization of AA to levopimaric acid (LA) following by Diels-Alder reactions between LA and MA. And then, a new bio-based polymeric thermoset was developed by the ring-opening polymerization of epoxidized soybean oil (ESO) with MPA catalyzed by 2-ethyl-4-methylimidazole (EMI)[4]. The curing was performed at 160℃ for 2h and 180℃ for 1h in a vacuum oven. The curing behaviors and the properties of the polymeric thermoset were studied with a comparison of a commercial curing agent, methylhexahydrophthalic anhydride (MHHPA). The epoxidized soybean oil cured by MPA can be used for various industrial applications not only due to its similar thermal and mechanical properties to that cured by the commercial curing agent but also the extra potential biodegradability

p A critical review of ferritin as a drug nanocarrier: Structure, properties, comparative advantages and challenges

Ferritin stores and releases iron ions in mammals. It is globally important as a drug nanocarrier. This is because of its unique hollow-spherical structure, desirable stability and biological properties. Novel drug-loading approaches plus various functionalization approaches have been developed to improve ferritin in response to differing demands in disease treatments. Here, we critically review ferritin drug delivery and evaluate its diverse drug-loading and functionalization approaches, we: (1) Introduce basic structural and property information related to ferritin as a drug nanocarrier; (2) Contrast in detail the different means to load drugs and the selection of drug loading means; (3) Discuss multiple ferritin functionalization approaches, together with related advantages and potential risks; and, (4) Compare ferritin with alternative, commonly-used drug nanocarriers. We conclude that despite that no drugs based on ferritin are commercially available, the market potential for it is significant, and evaluate future research directions. Findings from this work will be of immediate benefit and interest to a wide range of researchers and manufacturers for drug delivery using ferritin. (c) 2021 Published by Elsevier B.V. on behalf of Chinese Society of Particuology and Institute of Process Engineering, Chinese Academy of Sciences

J. Biotechnol.

Abstract - VII3-YP-027Jingxiu Bi, Zhiguo Su, Keith King, Anton Middelber

Cost-effective purification process development for chimeric hepatitis B core (HBc) virus-like particles assisted by molecular dynamic simulation

Inserting foreign epitopes to hepatitis B core (HBc) virus-like particles (VLPs) could influence the molecular conformation and therefore vary the purification process. In this study, a cost-effective purification process was developed for two chimeric HBc VLPs displaying Epstein-Barr nuclear antigens 1 (EBNA1), and hepatitis C virus (HCV) core. Both chimeric VLPs were expressed in soluble form with high production yields in Escherichia coli. Molecular dynamic (MD) simulation was employed to predict the stability of chimeric VLPs. HCV core-HBc was found to be less stable in water environment compared with EBNA1-HBc, indicating its higher hydrophobicity. Assisting with MD simulation, ammonium sulfate precipitation was optimized to remove host cell proteins with high target protein recovery yields. Moreover, 99% DNA impurities were removed using POROS 50 HQ chromatography. In characterization measurement, we found that inserting HCV core epitope would reduce the ratio of alpha-helix of HCV core-HBc. This could be another reason on the top of its higher hydrophobicity predicted by MD simulation, causing its less stability. Tertiary structure, transmission electron microscopy, and immunogenicity results indicate that two chimeric VLPs maintained correct VLP structure ensuring its bioactivity after being processed by the developed cost-effective purification approach

Intracellular microenvironment-responsive label-free autofluorescent nanogels for traceable gene delivery

Gene therapy presents a unique opportunity for the treatment of genetic diseases, but the lack of multifunctional delivery systems has hindered its clinical applications. Here, a new delivery vector, autofluorescent polyethyleneimine (PEI) nanogels, for highly efficient and traceable gene delivery is developed. Different from commercial high-molecular-weight PEI, the cationic nanogels are noncytotoxic and able to be fragmented due to their unique intracellular microenvironment-responsive structures. The biodegradable nanogels can effectively load plasmid DNA (pDNA), and the self-assembled polyplexes can be cleaved after cellular uptake to improve gene transfection efficiency. Most importantly, the nanogels and the nanogel/pDNA polyplexes are autofluorescent. The fluorescence is stable in blood plasma and responsive to the intracellular microenvironment. The breakup of the nanogels or polyplexes leads to the loss of fluorescence, and thus the gene delivery and carrier biodegradation processes can be monitored. The reported multifunctional system demonstrates excellent biocompatibility, high transfection efficiency, responsive biodegradability, controlled gene release, label-free and simultaneous fluorescence tracking, which will provide a new platform for future scientific investigation and practical implications in gene therapy

Design and Efficiency Research of a New Composite Vibrating Screen

A new composite vibrating mode is presented in this paper. Modeling and dynamic analysis are studied according to two-degree-of-freedom systems theory. The effects of vibration parameters, including swing angle, swing frequency, vibrating direction angle, and translation frequency, on the screening efficiency were researched by means of experiment research over a new laboratory-scale composite vibrating screen which is designed based on the new composite vibrating mode. The results are analysed in terms of curves and fitting equations. Compared to the translation mode and swing mode, the screening performance of the new composite vibrating mode, both in screening efficiency and in processing capacity, is significantly improved