High-level expression of a codon optimized recombinant dust mite allergen, Blo t 5, in Chinese hamster ovary cells.
Blo t 5 is a major allergen from house dust mite Blomia tropicalis. Purification of native Blo t 5 (nBlo t 5) from whole dust mite extract is tedious and gave low yield. In this study, we demonstrated that codon optimization facilitated high-level expression of Blo t 5 in Chinese hamster ovary (CHO)-K1 cells and thus allows production of sufficient recombinant cBlo t 5 for specific immunotherapy. A codon optimized Blo t 5 gene was synthesized by PCR and the codon optimized or wild-type Blo t 5 gene in pcDNA3.0 was transfected into CHO-K1 cells and stably selected with Geneticin (G418). Western-immunoblot analysis of spent culture media detected a positive band at 14kDa for the codon optimized but not wild-type gene transfectants. In addition, a stable CHO-K1 clone produced up to 13 mg/L of the cBlo t 5 protein having a high correlation of human IgE reactivities and allergenicity to the native Blo t 5, thus indicating proper conformation of this recombinant form.
Lim
L H
LH
Department of Paediatrics, National University of Singapore, Singapore.
Li
H Y
HY
Cheong
N
N
Lee
B W
BW
Chua
K Y
KY
D000485:Allergens / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D000818:Animals; D052179:Antigens, Plant; D016466:CHO Cells; D003062:Codon / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D005818:Genetic Engineering / Q000379:methods*; D008968:Molecular Conformation; D008970:Molecular Weight; D039981:Pyroglyphidae / Q000235:genetics* / Q000378:metabolism*; D011995:Recombination, Genetic; D014162:Transfection / Q000379:methods*
D000485:Allergens; D052179:Antigens, Plant; D003062:Codon; C094701:allergen Blo t 5