The biological activity of a recombinantly expressed (His)6-tagged peanut allergen (rAra h 1) is unaffected by endotoxin removal

Jensen, Louise Bjerremann; Torp, Anna Maria; Andersen, S. B.; Skov, Per Stahl; Poulsen, Lars K.; Knol, Edward F.; Hoffen, Els van

doi:10.1016/j.jim.2008.02.012

Cited by 19 publications

(22 citation statements)

References 9 publications

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“…Similarly, with the increase in the temperature effect, the micelle-poor phase showed a 2.40 times higher value of interference at 33 C. The increase observed may be associated with the presence of more molecules of Triton X-114 in the dilute phase (poor in micelles).…”

Section: Resultsmentioning

confidence: 80%

“…The systems were homogenized and equilibrated at 4 C, presenting a single phase. Subsequently, the systems were placed in a thermoregulated device, previously set at the desired temperature (29,33,37, and 41 C), and maintained there for 3 h to reach partition equilibrium. After that, the two coexisting micellar phases formed were withdrawn separately with great care, using syringe and needle sets, and the concentrations of GFPuv and LPS were determined.…”

Section: 62mentioning

confidence: 99%

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LPS removal from an E. coli fermentation broth using aqueous two‐phase micellar system

Lopes

Magalhães

Mazzola

et al. 2010

Biotechnology Progress

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In biotechnology, endotoxin (LPS) removal from recombinant proteins is a critical and challenging step in the preparation of injectable therapeutics, as endotoxin is a natural component of bacterial expression systems widely used to manufacture therapeutic proteins. The viability of large-scale industrial production of recombinant biomolecules of pharmaceutical interest significantly depends on the separation and purification techniques used. The aim of this work was to evaluate the use of aqueous two-phase micellar system (ATPMS) for endotoxin removal from preparations containing recombinant proteins of pharmaceutical interest, such as green fluorescent protein (GFPuv). Partition assays were carried out initially using pure LPS, and afterwards in the presence of E. coli cell lysate. The ATPMS technology proved to be effective in GFPuv recovery, preferentially into the micelle-poor phase (K(GFPuv) < 1.00), and LPS removal into the micelle-rich phase (%REM(LPS) > 98.00%). Therefore, this system can be exploited as the first step for purification in biotechnology processes for removal of higher LPS concentrations.

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Section: Resultsmentioning

confidence: 80%

Section: 62mentioning

confidence: 99%

LPS removal from an E. coli fermentation broth using aqueous two‐phase micellar system

Lopes

Magalhães

Mazzola

et al. 2010

Biotechnology Progress

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“…Samples were incubated at 4°C for 40 min on a rotor, then for 10 min at 37°C and centrifuged at 20,000 g for 20 min. After that, two liquid phases were visible and the upper one was recovered and treated again with the detergent under the same conditions for a total of three cycles [29,30]. LPS removal was confirmed by a quantitative and modified Limulus assay (Bio Whittaker, Walkersville, Md., USA), following the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

The Influence of Chitin on the Immune Response to the House Dust Mite Allergen Blo t 12

Zakzuk

Benedetti

Fernández-Caldas

et al. 2013

Int Arch Allergy Immunol

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Background: Information about the biological properties of Blomia tropicalis allergens is scarce. It is predicted that Blo t 12, an allergen with two described isoforms, contains a chitin-binding domain, similar to that found in peritrophins. Th2 adjuvant properties have been described for chitin. Therefore, it is feasible that binding to this carbohydrate influences its allergenicity. We aimed to evaluate the chitin-binding activity of Blo t 12 isoallergens and its effect on airway inflammation and antibody responses in a murine model of allergen sensitization. Methods: Chitin-binding assays were conducted with the recombinant isoallergens Blo t 12.0101 and Blo t 12.0102. BALB/c mice were sensitized via i.p. with any of the two isoforms (alone, with chitin or alum) and then challenged intranasally. Methacholine-induced bronchial hyperreactivity was tested by whole-body plethysmography and lung sections were stained with hematoxylin and eosin and periodic-acid Schiff. Total IgE and allergen-specific IgE, IgG1 and IgG2 levels were measured by ELISA. Results: The two isoforms bound chitin, but Blo t 12.0101 showed a stronger binding capacity. Both isoforms induced total and allergen-specific IgE, airway hyperreactivity, bronchial inflammation and mucus secretion without any adjuvant; however, when administered with chitin, Blo t 12.0101 induced higher total IgE levels. The IgG1/IgG2a ratio was significantly higher in mice immunized with Blo t 12.0101 than those immunized with Blo t 12.0102. As peritrophins, Blo t 12 was detected in mite feces. Conclusions: Blo t 12 isoforms are chitin-binding proteins that induce airway inflammation and bronchial hyperreactivity. However, for Blo t 12.0101, chitin reinforces its effects on total IgE production.

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“…The His-tagged recombinant Pen c 13 was bound to a Ni 2ϩ -chelate affinity column, and then endotoxin was removed by washing the resin in a centrifuge tube with binding buffer (20 mM Tris-HCl, pH 7.9, 0.5 M NaCl, 8 M urea, and 5 mM imidazole) containing 1% Triton X-114 (Sigma). The resin was loaded into a column and washed with binding buffer containing 0.1% Triton X-114 before elution of the recombinant protein with elution buffer (20 mM Tris-HCl, pH 7.9, 0.5 M NaCl, 8 M urea, and 60 mM imidazole) (24). The purified recombinant protein was denatured by heat at 95°C and called d-Pen c 13.…”

Section: Methodsmentioning

confidence: 99%

The Protease Allergen Pen c 13 Induces Allergic Airway Inflammation and Changes in Epithelial Barrier Integrity and Function in a Murine Model

Chen

Chuang

et al. 2011

Journal of Biological Chemistry

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Fungal allergens are associated with the development of asthma, and some have been characterized as proteases. Here, we established an animal model of allergic airway inflammation in response to continuous exposure to proteolytically active Pen c 13, a major allergen secreted by Penicillium citrinum. In functional analyses, Pen c 13 exposure led to increased airway hyperresponsiveness, significant inflammatory cell infiltration, mucus overproduction, and collagen deposition in the lung, dramatically elevated serum levels of total IgE and Pen c 13-specific IgE and IgG1, and increased production of the Th2 cytokines IL-4, IL-5, and IL-13 by splenocytes stimulated in vitro with Pen c 13. To examine the mechanisms involved in the regulation of allergenicity by Pen c 13, we performed two-dimensional fluorescence difference gel electrophoresis analysis combined with nano-LC-MS/MS, followed by bioinformatics analysis to identify potential targets that associated with allergic inflammation, which suggested that galectin-3 and laminin might be involved in novel pathogenic mechanisms. Finally, we focused on junctional proteins between cells, because, in addition to opening of the epithelial barrier by environmental proteases possibly being the initial step in the development of asthma, these proteins are also associated with actin rearrangement. Taken together, our findings indicate that Pen c 13 exposure causes junctional structure alterations and actin cytoskeletal rearrangements, resulting in increased permeability and airway structural changes. These effects probably change the lung microenvironment and foster the development of allergic sensitization.

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The biological activity of a recombinantly expressed (His)6-tagged peanut allergen (rAra h 1) is unaffected by endotoxin removal

Cited by 19 publications

References 9 publications

LPS removal from an E. coli fermentation broth using aqueous two‐phase micellar system

LPS removal from an E. coli fermentation broth using aqueous two‐phase micellar system

The Influence of Chitin on the Immune Response to the House Dust Mite Allergen Blo t 12

The Protease Allergen Pen c 13 Induces Allergic Airway Inflammation and Changes in Epithelial Barrier Integrity and Function in a Murine Model

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