Efficient soluble production of folded cat allergen Fel d 1 in Escherichia coli

Protein Expr Purif. 2021 Apr:180:105809. doi: 10.1016/j.pep.2020.105809. Epub 2020 Dec 15.

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.

Keywords: Allergen; Cytoplasmic oxidative folding; Disulfide bond formation; Escherichia coli; Fel d 1; Methionine sulfoxidation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Allergens* / biosynthesis
  • Allergens* / chemistry
  • Allergens* / genetics
  • Allergens* / isolation & purification
  • Animals
  • Cats
  • Glycoproteins* / biosynthesis
  • Glycoproteins* / chemistry
  • Glycoproteins* / genetics
  • Glycoproteins* / isolation & purification
  • Protein Folding*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification

Substances

  • Allergens
  • Glycoproteins
  • Recombinant Proteins
  • Fel d 1 protein, Felis domesticus