High-level expression in mammalian cells of recombinant house dust mite allergen ProDer p 1 with optimized codon usage

Int Arch Allergy Immunol. 2001 May;125(1):32-43. doi: 10.1159/000053794.

Abstract

Background: The major house dust mite allergen Der p 1 is associated with allergic diseases such as asthma. Production of recombinant Der p 1 was previously attempted, but with limited success. The present study describes the expression of recombinant (rec) ProDer p 1, a recombinant precursor form of Der p 1, in CHO cells.

Methods: As optimization of the codon usage may allow successful overexpression of protein in mammalian cells, a synthetic gene encoding ProDer p 1 was designed on the basis of the codon usage frequently found in highly expressed human genes. Gene synthesis was accomplished from a set of 14 mutually priming overlapping oligonucleotides and after two runs of polymerase chain reaction.

Results: COS cells transiently transfected with the synthetic ProDer p 1 gene produced up to 5--10 times as much ProDer p 1 compared with the expression level obtained after transfection with the authentic gene. To stably express the recombinant allergen, CHO-K1 cells were transfected with the ProDer p 1 synthetic gene, and one amplified recombinant clone produced up to 30 mg of recProDer p 1 per liter in the culture medium before purification. recProDer p 1 was secreted as an enzymatically inactive single-chain molecule presenting three glycosylated immunoreactive forms of 41, 38 and 36 kD. When examined with respect to direct binding, recProDer p 1 and natural Der p 1 displayed very similar IgE reactivities. However, IgE inhibition and histamine release assays showed a much higher reactivity to natural Der p 1 compared to recProDer p 1.

Conclusions: These data indicated that codon optimization represents an attractive strategy for high-level production of allergen in mammalian cells.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies, Blocking / immunology
  • Antigens, Dermatophagoides
  • Base Sequence
  • CHO Cells
  • Cells, Cultured
  • Codon
  • Cricetinae
  • Genes, Synthetic
  • Glycoproteins / biosynthesis
  • Glycoproteins / genetics*
  • Immunoglobulin E / immunology
  • Molecular Sequence Data
  • Protein Binding
  • Sequence Alignment
  • Sequence Homology
  • Transfection

Substances

  • Antibodies, Blocking
  • Antigens, Dermatophagoides
  • Codon
  • Glycoproteins
  • Immunoglobulin E