High-level expression in mammalian cells of recombinant house dust mite allergen ProDer p 1 with optimized codon usage.
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.
Massaer
M
M
Department of Applied Genetics, Institut de Biologie et de Médecine Moléculaires, Université Libre de Bruxelles, Gosselies, Belgium.
Mazzu
P
P
Haumont
M
M
Magi
M
M
Daminet
V
V
Bollen
A
A
Jacquet
A
A
D000595:Amino Acid Sequence; D000818:Animals; D019138:Antibodies, Blocking / Q000276:immunology; D039741:Antigens, Dermatophagoides; D001483:Base Sequence; D016466:CHO Cells; D002478:Cells, Cultured; D003062:Codon; D006224:Cricetinae; D005813:Genes, Synthetic; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D007073:Immunoglobulin E / Q000276:immunology; D008969:Molecular Sequence Data; D011485:Protein Binding; D016415:Sequence Alignment; D017385:Sequence Homology; D014162:Transfection
D019138:Antibodies, Blocking; D039741:Antigens, Dermatophagoides; D003062:Codon; D006023:Glycoproteins; D007073:Immunoglobulin E