Suspension‐cultured BY‐2 tobacco cells produce and mature immunologically active house dust mite allergens

Liénard, David; Dinh, Olivia Tran; Oort, Erika van; Overtvelt, Laurence Van; Bonneau, C; Wambre, Eric; Bardor, Muriel; Cosette, Pascal; Didierlaurent, A.; Borne, François Dorlhac de; Delon, R.; Ree, Ronald van; Moingeon, Philippe; Faye, Loı̈c; Gomord, Véronique

doi:10.1111/j.1467-7652.2006.00221.x

Cited by 39 publications

(26 citation statements)

References 53 publications

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“…Der p 2 has been produced successfully in various cell expression systems, including Escherichia coli, the yeast Saccharomyces cerevisiae and tobacco cells [11,12,13]. Der p 1 is a more complex protein expressed as a preproenzyme.…”

Section: Introductionmentioning

confidence: 99%

Recombinant Fusion Proteins Assembling Der p 1 and Der p 2 Allergens from <i>Dermatophagoides pteronyssinus</i>

Bussières

Floch²,

Bulder³

et al. 2010

Int Arch Allergy Immunol

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Background: Fusion proteins assembling multiple allergens can be engineered by recombinant DNA technologies in order to produce tools for diagnostic and immunotherapeutic purposes. Herein, we developed and characterized chimeras assembling Der p 1 and Der p 2 allergens as potential candidate vaccines against house dust mite allergy. Methods: Fusion proteins encompassing Der p 2 with either mature or proDer p 1 were expressed in Escherichia coli or Pichia pastoris. Forms with mutation in Der p 1 catalytic site were also engineered. Purified chimeras were characterized by immunoblotting, circular dichroism, disulfide bond mapping, basophil and T lymphocyte stimulation assays. Results: Four fusion proteins were expressed in E. coli as inclusion bodies, whereas only chimeras comprising proDer p 1 were obtained in yeast. All such hybrids formed polymers and aggregates, and yeast-expressed chimeras were unstable. Circular dichroism analysis performed after refolding of bacteria expressed chimeras encompassing mature Der p 1 confirmed partial folding, consistent with the occurrence of both correct and inappropriate intramolecular disulfide bonds. All fusion molecules were recognized by Der p 1- and Der p 2-specific human IgEs, monoclonal and polyclonal antibodies. Fusion proteins activate basophils from mite-allergic patients and trigger the proliferation of specific CD4+ T cells, albeit to a lower level when compared to individual allergens. Conclusions: Production of multiple Der p 1-Der p 2 fusion proteins exhibiting partial folding and proper antigenic properties has been achieved. Nonetheless, significant solubility and stability issues currently limit the application of such chimeras for immunotherapy or diagnostic.

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Section: Introductionmentioning

confidence: 99%

Recombinant Fusion Proteins Assembling Der p 1 and Der p 2 Allergens from <i>Dermatophagoides pteronyssinus</i>

Bussières

Floch²,

Bulder³

et al. 2010

Int Arch Allergy Immunol

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“…Other studies reported a successful expression of recombinant Der p 2 in E. coli , alone or as a fusion protein with glutathione-S-transferase or Der p 1 [29,37,49,50,51] as well as in eukaryotic hosts such as Saccharomyces cerevisiae [30,52] and tobacco cells [32]. Some of these molecules were found to resemble the natural counterpart in terms of antigenic properties.…”

Section: Discussionmentioning

confidence: 99%

“…Although Der p 2 has been previously produced in a recombinant form in a variety of expression systems [29,30,31,32], as of today, none of these studies has documented a bona fide natural conformation of the molecule to a level fulfilling the requirements for human administration. As Der p 2 exists as a variety of isoforms/variants [33,34,35], we first selected a prominent isoform following a mass spectrometry (MS) analysis of mite extracts.…”

Section: Introductionmentioning

confidence: 99%

Expression and Characterization of Natural-Like Recombinant Der p 2 for Sublingual Immunotherapy

Floch¹,

Bussières²,

Airouche³

et al. 2012

Int Arch Allergy Immunol

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Background: Recombinant allergens with a native conformation represent an alternative to natural extracts for immunotherapy and diagnostic purposes. Methods: We produced the Der p 2 mite allergen in Pichia pastoris and Escherichia coli. After purification by cation exchange chromatography, recombinant molecules were compared to their natural counterpart based upon structural (disulfide bonds, secondary structure, thermal stability) and immunological properties (antibody reactivity, basophil and T cell activation, tolerance induction in a murine sublingual immunotherapy model). Results: The Der p 2.0101 isoform was confirmed to be prevalent in Dermatophagoides pteronyssinus extracts. It was then produced as a secreted molecule in P. pastoris or refolded from E. coli inclusion bodies. The yeast-expressed rDer p 2 molecule exhibits a natural-like disulfide bridge distribution and secondary structure, whereas the E. coli-derived rDer p 2 presents some heterogeneity in cysteine bonds and a lower stability following thermal stress. The two recombinant as well as natural Der p 2 molecules exhibit comparable IgE recognition and activate basophil and CD4+ T cells. Sublingual immunotherapy of nDer p 2- sensitized mice using either one of the rDer p 2 molecules efficiently decreases airway hyperresponsiveness as well as Th2 responses. Conclusions: Natural and recombinant Der p 2 molecules produced in P. pastoris and E. coli exhibit comparable immunological properties despite distinct structural features. Natural-like cysteine pairing is a critical parameter to identify stable, well-folded and homogenous proteins appropriate for immunotherapy and diagnostic purposes.

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“…(Gomord et al 1997 ;Ma et al 2003 ;Chou and Shen 2010 ), directing the protein to the secretor pathway to collect it from the culture medium (e.g. : in in vitro cultures) (Hellwig et al 2004 ;Komarnytsky et al 2006 ;Lienard et al 2007 ;Benchabane et al 2008 ), co-expressing the protein with protease inhibitors (Faye et al 2005 ;Michaud et al 2005 ;Komarnytsky et al 2006 ), or expressing the recombinant protein as a fusion protein with a stable and highly expressed peptide (Nygren et al 1994 ;Stahl et al 1997 ).…”

Section: Plant Cell Levelmentioning

confidence: 99%

Molecular Farming in Plants

Álvarez

2014

Plant Biotechnology for Health

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Molecular farming can generally be defi ned as the production of molecules (proteins, fatty acids) for the pharmaceutical and chemical industries in transgenic organisms (plants, animals, etc.).Molecular farming in plants has advantageous aspects as biosecurity, they do not bear pathogens for humans or animals, and they do not produce toxins. Also, plant protein synthetic machinery is able to produce complex glycosylated proteins as they have a glycosylation pattern with slight difference respect of that of mammals, and can perform foldings.When displayed in in vitro conditions, molecular farming have the characteristic advantages of that type of culture, mainly the capacity of working under Good Manufactory practices as is required by the pharmaceutical industry. The critical aspects of plants for molecular farming are the different glycosylation patterns respect to mammals, the duration of the productive process, and the relatively low yields.The approval by the FDA of the fi rst medicine to be used in humans, taliglucerase alfa (ELELYSO®), has give an impulse to this technology but there are some drawbacks to be addressed, particularly related to low yields and regulatory aspects.

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Suspension‐cultured BY‐2 tobacco cells produce and mature immunologically active house dust mite allergens

Cited by 39 publications

References 53 publications

Recombinant Fusion Proteins Assembling Der p 1 and Der p 2 Allergens from <i>Dermatophagoides pteronyssinus</i>

Recombinant Fusion Proteins Assembling Der p 1 and Der p 2 Allergens from <i>Dermatophagoides pteronyssinus</i>

Expression and Characterization of Natural-Like Recombinant Der p 2 for Sublingual Immunotherapy

Molecular Farming in Plants

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