Separation of HIV‐1 gag virus‐like particles from vesicular particles impurities by hydroxyl‐functionalized monoliths

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.     

Self‐Assembled Cage‐Like Protein Structures

Proteins and protein‐based assemblies represent the most structurally and functionally diverse molecules found in nature. Protein cages, viruses and bacterial microcompartments are highly organized structures that are composed primarily of protein building blocks and play important roles in molecular ion storage, nucleic acid packaging and catalysis. The outer and inner surface of protein cages can be modified, either chemically or genetically, and the internal cavity can be used to template, store and arrange molecular cargo within a defined space. Owing to their structural, morphological, chemical and thermal diversity, protein cages have been investigated extensively for applications in nanotechnology, nanomedicine and materials science. Here we provide a concise overview of the most common icosahedral viral and nonviral assemblies, their role in nature, and why they are highly attractive scaffolds for the encapsulation of functional materials.     

Separation and quantification of double‐ and triple‐layered rotavirus‐like particles by CZE

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.     

Structure and function of cis-prenyl chain elongating enzymes

All carbon skeletons of isoprenoids, whose chain lengths vary widely from geranyl diphosphate (C10) to natural rubber (C>10,000), are synthesized by sequential condensation of isopentenyl diphosphate with an allylic diphosphate through catalytic functions of a group of enzymes commonly called "prenyltransferases." Prenyltransferases are classified into two major groups, trans- or (E)-prenyltransferases and cis- or (Z)-prenyltransferases, according to the geometry of the prenyl chain units in the products. From the year 1987, many genes encoding trans-prenyltransferases were cloned and clearly characterized. In contrast, the structure and detailed mechanism of cis-prenyltransferase was completely unknown until the identification of a gene encoding the undecaprenyl diphosphate (UPP) synthase from Micrococcus luteus B-P 26 in 1998. Not only the primary but also the tertiary structure of the UPP synthase is quite different from that of the trans-prenyltransferases. Multiple alignment of the primary structures of cis-prenyltransferases identified from various organisms reveals five highly conserved regions. Site-directed mutagenesis of the conserved amino acid residues in UPP synthases based on the crystal structure has elucidated the basic catalytic mechanisms. Moreover, comparison of the structures of short-, medium-, and long-chain cis-prenyltransferases reveals important amino acid residues for product chain length determination, which enabled us to understand the regulation mechanism of the ultimate chain length among cis-prenyltransferases.     

Building cavities in a fluid of spherical or rod-like particles: a contribution to the solvation free energy in isotropic and anisotropic polarizable continuum model

A general formalism for the calculation of cavitation energies in the framework of the scaled particle theory has been implemented in the Polarizable Continuum Model (PCM), contributing to the nonelectrostatic part of the molecular free energy in solution. The solute cavity and the solvent molecules are described as hard spherocylinders, whose radius and length are related to the actual molecular shape, while the solvent density is estimated from experimental data, or from the solvent molecular volume, suitably scaled. The present model can describe isotropic solutions of spherical and rod-like molecules in spherical or rod-like solvents, and also anisotropic solutions in which the solvent molecules are oriented in space: in this case, the cavitation energy also depends on the relative orientation of solute and solvent molecules. Test calculations have been performed on simple systems to evaluate the accuracy of the present approach, in comparison with other methods and with the available experimental estimates of the cavitation energy, giving encouraging results.     

Characterizing Methyl‐Bearing Side Chain Contacts and Dynamics Mediating Amyloid β Protofibril Interactions Using 13Cmethyl‐DEST and Lifetime Line Broadening

Many details pertaining to the formation and interactions of protein aggregates associated with neurodegenerative diseases are invisible to conventional biophysical techniques. We recently introduced ¹⁵N dark‐state exchange saturation transfer (DEST) and ¹⁵N lifetime line‐broadening to study solution backbone dynamics and position‐specific binding probabilities for amyloid β (Aβ) monomers in exchange with large (2–80 MDa) protofibrillar Aβ aggregates. Here we use ¹³C_methyl DEST and lifetime line‐broadening to probe the interactions and dynamics of methyl‐bearing side chains in the Aβ‐protofibril‐bound state. We show that all methyl groups of Aβ40 populate direct‐contact bound states with a very fast effective transverse relaxation rate, indicative of side‐chain‐mediated direct binding to the protofibril surface. The data are consistent with position‐specific enhancements of ¹³C_methyl‐${R{{{\rm tethered}\hfill \atop 2\hfill}}}$ values in tethered states, providing further insights into the structural ensemble of the protofibril‐bound state.     

Study of intact virus‐like particles of human papillomavirus by capillary electrophoresis

Virus‐like particles of human papillomavirus (HPV‐VLP), resulting from the self‐assembly of the capsid proteins (L1 or L1 and L2), have been widely used to study HPV as they are similar to the native virion. Moreover, two prophylactic vaccines, Gardasil^® and Cervarix^®, are based on HPV‐VLP L1. Analytical techniques currently used to characterize HPV‐VLP, such as SDS‐PAGE, Western blot, ELISA, are time‐consuming and semiquantitative. In this study, CE was evaluated for the analysis of intact HPV16‐VLP. The usefulness of capillary inner wall coating as well as various BGEs, pH, and detergent additives were investigated. Reproducible HPV‐VLP analysis in CE was achieved using poly(ethylene oxide)‐coated capillary and a BGE containing high salt concentration and low SDS concentration. The developed method enables HPV‐VLP detection in less than 10 min (migration times RSD: 1.6%). The identity of HPV‐VLP peak was confirmed by comparison with a sample obtained from a wild‐type baculovirus and with VLP‐based vaccine, Gardasil^®, after adjuvant dissolution. Finally, we applied the developed methodology to VLP‐based vaccines, demonstrating that CE could be successfully used for vaccine quality control.     

Computer simulation to investigate absorption of energies of arrayed disk‐like nanoiron particles under magnetic fields

In this research, we focus on the studying of absorbed energies of materials under an external magnetic field frequency of 0.5 GHz. This wave corresponds to microwave irradiation. The absorbent materials were arrayed disk‐like iron particles with dimension on the nanometer scale and magnetic responses of the particles were simulated by solving the Landau–Lifshitz–Gilbert equation. The external fields were applied from various directions and energies of absorption of the system were calculated. The maximum absorbed energies were found when the field was 135° ± 30° along the X‐axis or the Y‐axis. The current simulation demonstrated that the direction of applied field results in different absorption energies of the system.     

Building Proficient Enzymes with Foldamer Prostheses

Foldamers are non‐natural oligomers that adopt stable conformations reminiscent of those found in proteins. To evaluate the potential of foldameric subunits for catalysis, semisynthetic enzymes containing foldamer fragments constructed from α‐ and β‐amino acid residues were designed and characterized. Systematic variation of the α→β substitution pattern and types of β‐residue afforded highly proficient hybrid catalysts, thus demonstrating the feasibility of expanding the enzyme‐engineering toolkit with non‐natural backbones.     

Monodispersed submicron porous silica particles functionalized with CD derivatives for chiral CEC

Rapid and efficient enantioseparation of halogen aryl alcohols and β‐blockers propranolol and pindolol in packed bed CEC (p‐CEC) using as‐prepared submicron porous silica chiral stationary phases (CSPs) has been achieved. Monodispersed 0.66 and 0.81 μm chiral submicron porous silica spheres were prepared using tetramethoxysilane and hexadecyltrimethylammonium bromide, followed by a hydrothermal treatment method with ammonia–ethanol to expand the pore of silica spheres without changing their spherical morphology. A proper specific surface of ca. 230 m²/g and pore sizes average of 6–8 nm were obtained by this method. The submicron porous silica spheres were modified with mono‐6‐phenylcarbamoylated β‐CD via thiol‐en radical addition. They were packed into 9 cm 50 μm id capillary columns with photopolymerized monolithic frits. These submicron CSPs showed greater column efficiency (about 476 000 plates/m for 4‐iodophenyl‐1‐ethanol) and higher resolution than the corresponding 3 μm CSP.     

Mass spectrometric investigation of protein alkylation by the RNA footprinting probe kethoxal

The reactivity of the RNA footprinting reagent kethoxal (KT) toward proteins was investigated by electrospray ionization-Fourier transform mass spectrometry. Using standard peptides, KT was shown to selectively modify the guanidino group of arginine side chains at neutral pH, while primary amino groups of lysine and N-terminus were found to be unreactive under these conditions. Gas-phase fragmentation of KT adducts provided evidence for a cyclic 1,2-diol structure. Esterification of the 1,2-diol product was obtained in borate buffer, and its structure was also investigated by tandem mass spectrometry. When model proteins were probed with this RNA footprinting reagent, the adducts proved to be sufficiently stable to allow for the application of different peptide-mapping procedures to identify the location of modified arginines. Probing of proteins under native folding conditions provided modification patterns that very closely matched the structural context of arginines in the global protein structure. A strong correlation was demonstrated between the susceptibility to modification and residue accessibility calculated from the known 3D structure. When the complexes formed by HIV-1 nucleocapsid (NC) protein and RNA stemloops SL2 and SL3 were investigated, KT footprinting provided accurate information regarding the involvement of individual arginines in binding RNA and showed different reactivity according to their mode of interaction.     

Purification of recombinant adenovirus type 3 dodecahedric virus-like particles for biomedical applications using short monolithic columns

Adenovirus type 3 dodecahedric virus-like particles (Ad3 VLP) are an interesting delivery vector. They penetrate animal cells in culture very efficiently and up to 300,000 Ad3 VLP can be observed in one cell. The purification of such particles usually consists of several steps. In these work we describe the method development and optimization for the purification of Ad3 VLP using the Convective Interaction Media analytical columns (CIMac). Results obtained with the CIMac were compared to the already established two-step purification protocol for Ad3 VLP based on sucrose density gradient ultracentifugation and the Q-Sepharose ion-exchange column. Pure, concentrated and bioactive VLP were obtained and characterized by several analytical methods. The recovery of the Ad3 VLP was more than 50% and the purified fraction was almost completely depleted of DNA; less than 1% of DNA was present. The purification protocol was shortened from five days to one day and remarkably high penetration efficacy of the CIMac-purified vector was retained. Additionally, CIMac QA analytical column has proven to be applicable for the final and in-process control of various Ad3 VLP samples.     

The NICS (Nucleus-Independent Chemical Shift) as a probe of the relative stability of β-chalcogenovinylaldehydes stabilized through intramolecular chalcogen–chalcogen interactions

Density functional calculations, at the B3LYP/6-311+G(3df,2p) level, have been carried out for the complete series of β-chalcogenovinylaldehydes, CH(X)–CHCH–YH (X, Y=O, S, Se, Te), to estimate the strength of H–XY or XY–H intramolecular chalcogen–chalcogen interactions, through the use of appropriate homodesmotic reactions. For the same set of compounds the value of the nucleus-independent chemical shift (NICS), on points 1 Å above the corresponding ring critical point, has been obtained at the B3LYP/6-311+G(3df,2p) level. For non-stabilizing chalcogen–chalcogen interactions the NICS value is positive, while the opposite is found when the interaction is stabilizing. In general, there is a good linear correlation between both magnitudes and therefore, we can conclude that NICS value is a reliable probe of the strength of intramolecular chalcogen–chalcogen interactions in this set of compounds.     

A Redox‐Dependent Electrochromic Material: Tetri‐EDOT Substituted Thieno[3,2‐b]thiophene

Organic electrochromic materials change color rapidly under applied potential. A butterfly‐shaped compound, 5,5′,‐5″,‐5′″‐(thieno[3,2‐b]thiophene‐2,3,5,6‐tetrayl) tetrakis‐(2,3‐dihydrothieno[3,4‐b][1,4]dioxine) (t‐EDOT‐TT) is synthesized for the first time and polymerized at different potentials via electropolymerization technique. By applying different polymerization potentials, the optical and electrochromic properties of this newly synthesized polymer can be tuned. Owing to the dependence of functional group position in the polymer structure on the redox potential, this polymer can be utilized in very interesting organic optoelectronic applications.