scholarly journals A Four-Step Purification Process for Gag VLPs: From Culture Supernatant to High-Purity Lyophilized Particles

Vaccines ◽  
2021 ◽  
Vol 9 (10) ◽  
pp. 1154
Author(s):  
Irene González-Domínguez ◽  
Elianet Lorenzo ◽  
Alice Bernier ◽  
Laura Cervera ◽  
Francesc Gòdia ◽  
...  
Keyword(s):  
Membrane Filtration ◽  
Structural Integrity ◽  
Culture Media ◽  
Exchange Chromatography ◽  
Cell Culture Media ◽  
Hek 293 ◽  
Host Cell Proteins ◽  
Double Stranded Dna ◽  
293 Cells ◽  
Delivery Strategies

Gag-based virus-like particles (VLPs) have high potential as scaffolds for the development of chimeric vaccines and delivery strategies. The production of purified preparations that can be preserved independently from cold chains is highly desirable to facilitate distribution and access worldwide. In this work, a nimble purification has been developed, facilitating the production of Gag VLPs. Suspension-adapted HEK 293 cells cultured in chemically defined cell culture media were used to produce the VLPs. A four-step downstream process (DSP) consisting of membrane filtration, ion-exchange chromatography, polishing, and lyophilization was developed. The purification of VLPs from other contaminants such as host cell proteins (HCP), double-stranded DNA, or extracellular vesicles (EVs) was confirmed after their DSP. A concentration of 2.2 ± 0.8 × 109 VLPs/mL in the lyophilized samples was obtained after its storage at room temperature for two months. Morphology and structural integrity of purified VLPs was assessed by cryo-TEM and NTA. Likewise, the purification methodologies proposed here could be easily scaled up and applied to purify similar enveloped viruses and vesicles.

scholarly journals Structure of the omalizumab Fab

2015 ◽  
Vol 71 (4) ◽  
pp. 419-426 ◽  
Author(s):  
Rasmus K. Jensen ◽  
Melanie Plum ◽  
Luna Tjerrild ◽  
Thilo Jakob ◽  
Edzard Spillner ◽  
...  
Keyword(s):  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Structural Basis ◽  
Hek 293 ◽  
Functional Studies ◽  
293 Cells ◽  
Exclusion Chromatography ◽  
Ige Mediated ◽  
High Affinity Ige Receptor

Omalizumab is a humanized anti-IgE antibody that inhibits the binding of IgE to its receptors on mast cells and basophils, thus blocking the IgE-mediated release of inflammatory mediators from these cells. Omalizumab binds to the Fc domains of IgE in proximity to the binding site of the high-affinity IgE receptor Fc∊RI, but the epitope and the mechanisms and conformations governing the recognition remain unknown. In order to elucidate the molecular mechanism of its anti-IgE activity, the aim was to analyse the interaction of omalizumab with human IgE. Therefore, IgE Fc C∊2–4 was recombinantly produced in mammalian HEK-293 cells. Functionality of the IgE Fc was proven by ELISA and mediator-release assays. Omalizumab IgG was cleaved with papain and the resulting Fab was purified by ion-exchange chromatography. The complex of IgE Fc with omalizumab was prepared by size-exclusion chromatography. However, crystals containing the complex were not obtained, suggesting that the process of crystallization favoured the dissociation of the two proteins. Instead, two structures of the omalizumab Fab with maximum resolutions of 1.9 and 3.0 Å were obtained. The structures reveal the arrangement of the CDRs and the position of omalizumab residues known from prior functional studies to be involved in IgE binding. Thus, the structure of omalizumab provides the structural basis for understanding the function of omalizumab, allows optimization of the procedure for complex crystallization and poses questions about the conformational requirements for anti-IgE activity.

scholarly journals Plasticity of the HEK-293 cells, related to the culture media, as platform to produce a subunit vaccine against classical swine fever virus

AMB Express ◽  
2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Elianet Lorenzo ◽  
Lidice Méndez ◽  
Elsa Rodríguez ◽  
Nemecio Gonzalez ◽  
Gleysin Cabrera ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Subunit Vaccine ◽  
Culture Media ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
A Subunit

scholarly journals Modulation of circulatory residence of recombinant acetylcholinesterase through biochemical or genetic manipulation of sialylation levels

1998 ◽  
Vol 336 (3) ◽  
pp. 647-658 ◽  
Author(s):  
Theodor CHITLARU ◽  
Chanoch KRONMAN ◽  
Menachem ZEEVI ◽  
Michal KAM ◽  
Adrian HAREL ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Anion Exchange ◽  
Acid Content ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Sialic Acid Content ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
High Level

Sialylation of N-glycans associated with recombinant human acetylcholinesterase (rHuAChE) has a central role in determining its circulatory clearance rate. Human embryonal kidney 293 (HEK-293) cells, which are widely used for the expression of recombinant proteins, seem to be limited in their ability to sialylate overexpressed rHuAChE. High-resolution N-glycan structural analysis, by gel permeation, HPLC anion-exchange chromatography and high-pH anion-exchange chromatography (HPAEC), revealed that the N-glycans associated with rHuAChE produced in HEK-293 cells belong mainly to the complex-biantennary class and are only partly sialylated, with approx. 60% of the glycans being monosialylated. This partial sialylation characterizes rHuAChE produced by cells selected for high-level expression of the recombinant protein. In low-level producer lines, the enzyme exhibits a higher sialic acid content, suggesting that undersialylation of rHuAChE in high-level producer lines stems from a limited endogenous glycosyltransferase activity. To improve sialylation in HEK-293 cells, rat liver β-galactoside α-2,6-sialyltransferase cDNA was stably transfected into cells expressing high levels of rHuAChE. rHuAChE produced by the modified cells displayed a significantly higher proportion of fully sialylated glycans as shown by sialic acid incorporation assays, direct measurement of sialic acid, and HPAEC glycan profiling. Genetically modified sialylated rHuAChE exhibited increased circulatory retention (the slow-phase half-life, t½β, was 130 min, compared with 80 min for the undersialylated enzyme). Interestingly, the same increase in circulatory residence was observed when rHuAChE was subjected to extensive sialylation in vitro. The engineered HEK-293 cells in which the glycosylation machinery was modified might represent a valuable tool for the high level of expression of recombinant glycoproteins whose sialic acid content is important for their function or for pharmacokinetic behaviour.

scholarly journals In Situ Swelling Behavior of Chitosan-Polygalacturonic Acid/Hydroxyapatite Nanocomposites in Cell Culture Media

2010 ◽  
Vol 2010 ◽  
pp. 1-12 ◽  
Author(s):  
Rohit Khanna ◽  
Kalpana S. Katti ◽  
Dinesh R. Katti
Keyword(s):  
Cell Culture ◽  
Structural Integrity ◽  
Culture Media ◽  
Sample Surface ◽  
Nanocomposite Films ◽  
Apatite Formation ◽  
Polygalacturonic Acid ◽  
Cell Culture Media ◽  
Polyelectrolyte Complexes

The molecular and mechanical characteristics of in situ degradation behavior of chitosan-polygalacturonic acid/hydroxyapatite (Chi-PgA-HAP) nanocomposite films is investigated using Fourier Transform Infrared spectroscopy (FTIR), Atomic Force Microscopy (AFM), and modulus mapping techniques for up to 48 days of soaking in cell culture media. The surface molecular structure of media-soaked samples changes over the course of 48 days of soaking, as indicated by significant changes in phosphate vibrations (1200–900 ) indicating apatite formation. Chitosan-Polygalacturonic acid polyelectrolyte complexes (PECs) govern structural integrity of Chi-PgA-HAP nanocomposites and FTIR spectra indicate that PECs remain intact until 48 days of soaking. In situ AFM experiments on media-soaked samples indicate that soaking results in a change in topography and swelling proceeds differently at the initial soaking periods of about 8 days than for longer soaking. In situ modulus mapping experiments are done on soaked samples by probing 1–3 nm of surface indicating elastic moduli of 4 GPa resulting from proteins adsorbed on Chi-PgA-HAP nanocomposites. The elastic modulus decreases by 2 GPa over a long exposure to cell culture media (48 days). Thus, as water enters the Chi-PgA-HAP sample, surface molecular interactions in Chi-PgA-HAP structure occur that result in swelling, causing small changes in nanoscale mechanical properties.

High-pressure freezing of cells in suspension for low-voltage scanning electron microscopy (LVSEM)

1991 ◽  
Vol 49 ◽  
pp. 64-65
Author(s):  
Marek Malecki ◽  
James Pawley ◽  
Hans Ris
Keyword(s):  
Electron Microscopy ◽  
High Pressure ◽  
Low Voltage ◽  
Culture Media ◽  
Cell Structure ◽  
Freeze Substitution ◽  
Ice Crystal ◽  
High Pressure Freezing ◽  
Cell Culture Media ◽  
Voltage Scanning

The ultrastructure of cells suspended in physiological fluids or cell culture media can only be studied if the living processes are stopped while the cells remain in suspension. Attachment of living cells to carrier surfaces to facilitate further processing for electron microscopy produces a rapid reorganization of cell structure eradicating most traces of the structures present when the cells were in suspension. The structure of cells in suspension can be immobilized by either chemical fixation or, much faster, by rapid freezing (cryo-immobilization). The fixation speed is particularly important in studies of cell surface reorganization over time. High pressure freezing provides conditions where specimens up to 500μm thick can be frozen in milliseconds without ice crystal damage. This volume is sufficient for cells to remain in suspension until frozen. However, special procedures are needed to assure that the unattached cells are not lost during subsequent processing for LVSEM or HVEM using freeze-substitution or freeze drying. We recently developed such a procedure.

Structural integrity after cold storage of human epidermal model in hypothermosol: A correlative ultrastructural and fluorescent assay

1994 ◽  
Vol 52 ◽  
pp. 270-271
Author(s):  
Henry H. Eichelberger ◽  
John G. Baust ◽  
Robert G. Van Buskirk
Keyword(s):  
Cold Storage ◽  
Structural Integrity ◽  
Culture Media ◽  
Cell Structure ◽  
Natural State ◽  
Sodium Cacodylate ◽  
Ruthenium Tetroxide ◽  
Cacodylate Buffer ◽  
Before And After

For research in cell differentiation and in vitro toxicology it is essential to provide a natural state of cell structure as a benchmark for interpreting results. Hypothermosol (Cryomedical Sciences, Rockville, MD) has proven useful in insuring the viability of synthetic human epidermis during cold-storage and in maintaining the epidermis’ ability to continue to differentiate following warming.Human epidermal equivalent, EpiDerm (MatTek Corporation, Ashland, MA) consisting of fully differentiated stratified human epidermal cells were grown on a microporous membrane. EpiDerm samples were fixed before and after cold-storage (4°C) for 5 days in Hypothermosol or skin culture media (MatTek Corporation) and allowed to recover for 7 days at 37°C. EpiDerm samples were fixed 1 hour in 2.5% glutaraldehyde in sodium cacodylate buffer (pH 7.2). A secondary fixation with 0.2% ruthenium tetroxide (Polysciences, Inc., Warrington, PA) in sodium cacodylate was carried out for 3 hours at 4°C. Other samples were similarly fixed, but with 1% Osmium tetroxide in place of ruthenium tetroxide. Samples were dehydrated through a graded acetone series, infiltrated with Spurrs resin (Polysciences Inc.) and polymerized at 70°C.

Cell culture media: NMR approaches to evaluating quality and nutrient consumption

Planta Medica ◽  
2016 ◽  
Vol 81 (S 01) ◽  
pp. S1-S381
Author(s):  
KB Killday ◽  
AS Freund ◽  
C Fischer ◽  
KL Colson
Keyword(s):  
Cell Culture ◽  
Culture Media ◽  
Cell Culture Media ◽  
Nutrient Consumption

The Influence of Glycosylation on the Catalytic and Fibrinolytic Properties of Pro-Urokinase

1992 ◽  
Vol 68 (05) ◽  
pp. 539-544 ◽  
Author(s):  
Catherine Lenich ◽  
Ralph Pannell ◽  
Jack Henkin ◽  
Victor Gurewich
Keyword(s):  
Escherichia Coli ◽  
Mammalian Cell ◽  
Human Gene ◽  
Culture Media ◽  
Mammalian Cell Culture ◽  
Glycosylation Site ◽  
Clot Lysis ◽  
Cell Culture Media ◽  
E Coli ◽  
The Uk

SummaryWe previously found that human pro-UK expressed in Escherichia coli is more active in fibrinolysis than recombinant human pro-UK obtained from mammalian cell culture media. To determine whether this difference is related to the lack of glycosylation of the E. coli product, we compared the activity of E. coli-derived pro-UK [(-)pro-UK] with that of a glycosylated pro-UK [(+)pro-UK] and of a mutant of pro-UK missing the glycosylation site at Asn-302 [(-) (302) pro-UK]. The latter two pro-UKs were obtained by expression of the human gene in a mammalian cell. The nonglycosylated pro-UKs were activated by plasmin more efficiently (≈2-fold) and were more active in clot lysis (1.5-fold) than the (+)pro-UK. Similarly, the nonglycosylated two-chain derivatives (UKs) were more active against plasminogen and were more rapidly inactivated by plasma inhibitors than the (+)UK.These findings indicate that glycosylation at Asn-302 influences the activity of pro-UK/UK and could be the major factor responsible for the enhanced activity of E. coli-derived pro-UK.

scholarly journals Species-Specific Regulation of TRPM2 by PI(4,5)P2 via the Membrane Interfacial Cavity

2021 ◽  
Vol 22 (9) ◽  
pp. 4637
Author(s):  
Daniel Barth ◽  
Andreas Lückhoff ◽  
Frank J. P. Kühn
Keyword(s):  
Amino Acid Residues ◽  
Hek 293 ◽  
Complete Inactivation ◽  
293 Cells ◽  
Activation Mechanisms ◽  
Specific Regulation ◽  
Species Specific ◽  
Inside Out ◽  
Positively Charged

The human apoptosis channel TRPM2 is stimulated by intracellular ADR-ribose and calcium. Recent studies show pronounced species-specific activation mechanisms. Our aim was to analyse the functional effect of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), commonly referred to as PIP2, on different TRPM2 orthologues. Moreover, we wished to identify the interaction site between TRPM2 and PIP2. We demonstrate a crucial role of PIP2, in the activation of TRPM2 orthologues of man, zebrafish, and sea anemone. Utilizing inside-out patch clamp recordings of HEK-293 cells transfected with TRPM2, differential effects of PIP2 that were dependent on the species variant became apparent. While depletion of PIP2 via polylysine uniformly caused complete inactivation of TRPM2, restoration of channel activity by artificial PIP2 differed widely. Human TRPM2 was the least sensitive species variant, making it the most susceptible one for regulation by changes in intramembranous PIP2 content. Furthermore, mutations of highly conserved positively charged amino acid residues in the membrane interfacial cavity reduced the PIP2 sensitivity in all three TRPM2 orthologues to varying degrees. We conclude that the membrane interfacial cavity acts as a uniform PIP2 binding site of TRPM2, facilitating channel activation in the presence of ADPR and Ca2+ in a species-specific manner.

Sign in / Sign up

Export Citation Format

Share Document