Direct expression of Der f2, a major house dust mite allergen, in Escherichia coli.
Der f2 protein in a highly antigenic form was directly expressed in bacteria. Plasmid pFLU11 derived from pKK233-2 was designed to express methionyl-Der f2 under the control of the trc promoter and it has the replication origin of pUC118 instead of its original to increase the copy number. This expression plasmid directed the synthesis of recombinant Der f2 (rDer f2) protein in an insoluble form of inclusion bodies in Escherichia coli cells. The high copy number plasmid pFLU11 conferred the efficient production of the Der f2 protein in E. coli, when compared to a nonchanged origin material. rDer f2 inclusion bodies were easily solubilized in urea and renatured by dialysis to assume the active form. The rDer f2 protein was purified by means of anion exchange and gel filtration chromatography. This expression system yielded about 10 mg of purified rDer f2 protein from the 1L culture. Purified rDer f2 protein reacted with IgE from patient sera almost identically to the native Der f2 in the RAST enzyme immunoassay and skin prick test.
Iwamoto
N
N
Central Research Laboratory, Asahi Breweries, Tokyo, Japan.
Nishiyama
C
C
Yasuhara
T
T
Saito
A
A
Yuuki
T
T
Okumura
Y
Y
Okudaira
H
H
D000485:Allergens / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D000818:Animals; D039741:Antigens, Dermatophagoides; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D006969:Hypersensitivity, Immediate / Q000276:immunology; D007073:Immunoglobulin E / Q000378:metabolism; D066298:In Vitro Techniques; D008925:Mites / Q000235:genetics* / Q000276:immunology*; D015394:Molecular Structure; D010957:Plasmids / Q000235:genetics; D011852:Radioallergosorbent Test; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology
D000485:Allergens; D039741:Antigens, Dermatophagoides; D006023:Glycoproteins; D011994:Recombinant Proteins; D007073:Immunoglobulin E