Search Results (5)

Antibodies are complex protein structures, and producing them using eukaryotic expression systems presents significant challenges. One frequently overlooked aspect of expression vectors is the nucleotide sequence encoding the signal peptide, which plays a pivotal role in facilitating the secretion of recombinant proteins. This study presents the development of an integrative vector, pVEAL3, for expressing full-length recombinant monoclonal antibodies in mammalian cells. The vector features a distinctive nucleotide sequence that encodes an artificial chimeric signal peptide with the following amino acid sequence: MMRTLILAVLLVYFCATVHC. Additionally, the vector incorporates several regulatory elements to enhance antibody expression, including the Gaussia luciferase signal sequence, internal ribosome entry site (IRES), P2A peptide, and a furin cleavage site. These elements coordinate to regulate the synthesis levels of the antibody chains. The analysis of clones obtained via transfection with the developed vector showed that over 95% of them secreted antibodies at levels significantly higher than those of the control. The immunochemical analysis of the chimeric antibody produced by the CHO-K1-10H10ch cell line confirmed the preservation of its functional activity. Full article

(1) Recombinant protein production in mammalian cells is either based on transient transfection processes, often inefficient and underlying high batch-to-batch variability, or on laborious generation of stable cell lines. Alternatively, BacMam, a transduction process using the baculovirus, can be employed. (2) Six transfecting agents were compared to baculovirus transduction in terms of transient and stable protein expression characteristics of the model protein ACE2-eGFP using HEK293-6E, CHO-K1, and Vero cell lines. Furthermore, process optimization such as expression enhancement using sodium butyrate and TSA or baculovirus purification was assessed. (3) Baculovirus transduction efficiency was superior to all transfection agents for all cell lines. Transduced protein expression was moderate, but an 18-fold expression increase was achieved using the enhancer sodium butyrate. Ultracentrifugation of baculovirus from a 3.5 L bioreactor significantly improved the transduction efficiency and protein expression. Stable cell lines were obtained with each baculovirus transduction, yet stable cell line generation after transfection was highly unreliable. (4) This study demonstrated the superiority of the BacMam platform to standard transfections. The baculovirus efficiently transduced an array of cell lines both transiently and stably and achieved the highest efficiency for all tested cell lines. The feasibility of the scale-up of baculovirus production was demonstrated and the possibility of baculovirus purification was successfully explored. Full article

The cell-surface topography and density of nicotinic acetylcholine receptors (nAChRs) play a key functional role in the synapse. Here we employ in parallel two labeling and two super-resolution microscopy strategies to characterize the distribution of this receptor at the plasma membrane of the mammalian clonal cell line CHO-K1/A5. Cells were interrogated with two targeted techniques (confocal microscopy and stimulated emission depletion (STED) nanoscopy) and single-molecule nanoscopy (stochastic optical reconstruction microscopy, STORM) using the same fluorophore, Alexa Fluor 647, tagged onto either α-bungarotoxin (BTX) or the monoclonal antibody mAb35. Analysis of the topography of nanometer-sized aggregates (“nanoclusters”) was carried out using STORMGraph, a quantitative clustering analysis for single-molecule localization microscopy based on graph theory and community detection, and ASTRICS, an inter-cluster similarity algorithm based on computational geometry. Antibody-induced crosslinking of receptors resulted in nanoclusters with a larger number of receptor molecules and higher densities than those observed in BTX-labeled samples. STORM and STED provided complementary information, STED rendering a direct map of the mesoscale nAChR distribution at distances ~10-times larger than the nanocluster centroid distances measured in STORM samples. By applying photon threshold filtering analysis, we show that it is also possible to detect the mesoscale organization in STORM images. Full article

Atmospheric cold plasma (ACP) is under investigation for an extensive range of biocontrol applications in food biosystems. However, the development of a novel intervention technology requires a thorough evaluation of the potential for negative effects and the implications for the human and animal food chains’ safety. The evaluations were performed using a contained, high-voltage, dielectric barrier discharge plasma system. The cytotoxicity of two types of food models—a liquid model (wheat model medium (WMM)) vs. a solid model (wheat grain extract (WGE)) was compared in vitro using the mammalian cell line CHO-K1. The residual toxicity of ACP treatment of grains for food purposes was assessed using the invertebrate model Tribolium castaneum, by feeding the beetles with flour produced from ACP-treated wheat grains. The cytotoxic effects and changes in the chemistry of the ACP-treated samples were more pronounced in samples treated in a liquid form as opposed to actual wheat grains. The feeding trial using T. castaneum demonstrated no negative impacts on the survivability or weight profiles of insects. Investigations into the interactions of plasma-generated species with secondary metabolites in the food matrices are necessary to ensure the safety of plasma for food applications. Full article

Zinc Oxide (ZnO) nanoparticles are suspected to produce toxic effects toward mammalian cells; however, discrepancies in the extent of this effect have been reported between different cell lines. Simultaneously, high levels of ultraviolet (UV-C) radiation can have carcinogenic effects. The mechanism of this effect is also not well understood. Due to similarities in phenotype morphology after cell exposure to ZnO nanoparticles and UV-C irradiation, we emit the hypothesis that the toxicity of both these factors is related to damage of cellular membranes and affect their sterol content. Wild-type Chinese Hamster Ovary (CHO-K1) cells were exposed to ZnO nanoparticles or UV-C radiation. The amount of absorbed ZnO was determined by UV-visible spectroscopy and the changes in sterol profiles were evaluated by gas chromatography. Cell viability after both treatments was determined by microscopy. Comparing morphology results suggested similarities in toxicology events induced by ZnO nanoparticles and UV exposure. UV-C exposure for 360 min disrupts the sterol metabolic pathway by increasing the concentration of cholesterol by 21.6-fold. This increase in cholesterol production supports the hypothesis that UV irradiation has direct consequences in initiating sterol modifications in the cell membrane. Full article