Abstract
The major cat allergen Fel d 1 is one of the most common and potent causes of animal related allergy. Medical treatment of cat allergy has relied on immunotherapy carried out with cat dander extract. This approach has been problematic, mainly due to inconsistent levels of the major allergen in the produced extracts. Recombinant DNA technology has been proposed as an alternative method to produce more consistent pharmaceuticals for immunotherapy and diagnostics of allergy. Current approaches to produce recombinant Fel d 1 (recFel d 1) in the cytoplasm of Escherichia coli have however resulted in protein folding deficiencies and insoluble inclusion body formation, requiring elaborate in vitro processing to acquire folded material. In this study, we introduce an efficient method for cytoplasmic production of recFel d 1 that utilizes eukaryotic folding factors to aid recFel d 1 to fold and be produced in the soluble fraction of E. coli. The solubly expressed recFel d 1 is shown by biophysical in vitro experiments to contain structural disulfides, is extremely stable, and has a sensitivity for methionine sulfoxidation. The latter is discussed in the context of functional relevance.
Highlights
Exposure to proteins from other species, either airborne or delivered via food or injection, results in an immune response in humans that may lead to allergic sensitization with symptoms ranging from discomforting rhinitis to life-threatening asthmatic inflammatory disease
We show that recFel d 1 can be produced solubly in the cytoplasm using CyDisCo with three disulfides and an extremely stable fold
Purification from inclusion bodies typically requires solubilization with chaotropic agents, in vitro refolding, and elaborate purification procedures to obtain homogenously folded soluble allergen. This labo rious approach limits the applicability of E. coli as an efficient expression host for large scale protein production
Summary
Exposure to proteins from other species, either airborne or delivered via food or injection, results in an immune response in humans that may lead to allergic sensitization with symptoms ranging from discomforting rhinitis to life-threatening asthmatic inflammatory disease. The classical method to treat allergy is allergen-specific immunotherapy (SIT). Airborne protein allergens from domestic cat (Felis domesticus) are among the most common and potent causes of allergy worldwide [2]. The major cat allergen, Fel d 1, is known to cause an immune response for more than 90% of cat allergy patients [3]. Two different recombinant Fel d 1 (recFel d 1) con structs have been successfully produced as inclusion bodies in Escher ichia coli and refolded to their native state [4,5]. While the natural Fel d 1 (nFel d 1) is a tetramer of two heterodimers, in the recFel d 1 molecules, the chains are typically fused together (Fig. 1A). Either recFel d 1 fusion protein could act as an alternative allergen source for SIT
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