Structural and immunological characterization of recombinant ovomucoid expressed in Escherichia coli.
The expression of recombinant allergens is becoming new insights of an important diagnosis and the therapy of allergies as well as molecular approaches to immunological and structural studies of allergens. Ovomucoid is a major food allergens in the hen's egg white which causes immediate food-hypersensitivity reactions mainly in children. A gene coding for the cDNA representing an entire ovomucoid molecule has been cloned in Escherichia coli under the control of T5 promoter fused with six-Histidine tag at the amino terminal end. Upon induction, the E. coli cells, harbouring this construct, expressed the recombinant protein as a soluble fraction and the recombinant ovomucoid protein was purified to electrophoeretic homogeneity using Ni2+ nitrilotriacetic acid agarose affinity chromatography. Immunoblot analysis showed that human IgE and IgG binding activities of the recombinant ovomucoid was identical to that of native analogue. The antigenicity and allergenicity of recombinant ovomucoid were almost same as that of native form when tested with an ELISA using six individual patient's serum. CD spectra indicated that that the recombinant ovomucoid has more alpha-helix and less beta-structure than native form. These results show that the recombinant ovomucoid constructed in this study could be used for further studies on the immunological and structural studies of ovomucoid.
Rupa
Prithy
P
Department of Food Science, University of Guelph Guelph, Ontario N1G 2W1, Canada.
Mine
Yoshinori
Y
D000485:Allergens / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D000818:Animals; D002645:Chickens; D003001:Cloning, Molecular; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology*; D007074:Immunoglobulin G / Q000276:immunology*; D008970:Molecular Weight; D010059:Ovomucin / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology* / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D013329:Structure-Activity Relationship
D000485:Allergens; D000939:Epitopes; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D010059:Ovomucin; D007073:Immunoglobulin E