Expression, purification and immunochemical characterization of recombinant bovine beta-lactoglobulin, a major cow milk allergen.
The immunological characteristics of a recombinant beta-lactoglobulin were studied using monoclonal antibodies, polyclonal antiserum and sera from allergic patients. Recombinant beta-lactoglobulin (rBLG) was expressed in Escherichia coli strain DH5alpha and purified as described previously [Cho et al. (1994) J. Biol. Chem. 269, 11 102-11 107]. The method has been modified by adding an immunoaffinity purification step. A quantity of 5-10mg of purified rBLG per liter of medium culture can be produced. rBLG shared the same molecular weight as the natural BLG (nBLG) and also possessed at least one intrachain disulfide bridge. In HPLC, rBLG appeared as a single peak, and the purity was estimated to be greater than 95%. All the monoclonal antibodies (mAbs) used in this study recognized different epitopes of the BLG and presented compatible binding. No differences could be detected between rBLG and nBLG when tested in a Western blot with rabbit polyclonal antiserum or with three mAbs that bound preferentially the reduced and S-carboxymethylated form of BLG. In a competitive enzyme immunoassay (EIA) using either a rabbit polyclonal antiserum or four mAbs that recognized conformational epitopes, we could not distinguish between rBLG or nBLG. In direct ELISA using nBLG or rBLG as the immobilized allergen, we measured a similar concentration of specific anti-BLG IgE in five sera from allergic patients. The results of this study indicate that we have obtained a rBLG with biochemical and immunological properties very similar to nBLG.
Chatel
J M
JM
INRA-CEA, DRM-SPI, Gif Sur Yvette, France.
Bernard
H
H
Clement
G
G
Frobert
Y
Y
Batt
C A
CA
Gavalchin
J
J
Peltres
G
G
Wal
J M
JM
D000485:Allergens / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001666:Binding Sites, Antibody / Q000276:immunology; D002417:Cattle; D007120:Immunochemistry; D007073:Immunoglobulin E / Q000276:immunology; D007782:Lactoglobulins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*
D000485:Allergens; D000911:Antibodies, Monoclonal; D007782:Lactoglobulins; D011994:Recombinant Proteins; D007073:Immunoglobulin E