Recombinant barley-produced antibody for detection and immunoprecipitation of the major bovine milk allergen, β-lactoglobulin.
Recombinant allergens and antibodies are needed for diagnostic, therapeutic, food processing and quality verification purposes. The aim of this work was to develop a barley-based production system for β-lactoglobulin (BLG) specific immunoglobulin E antibody (D1 scFv). The expression level in the best barley cell clone was 0.8-1.2 mg/kg fresh weight, and was constant over an expression period of 21 days. In the case of barley grains, the highest stable productivity (followed up to T2 grains) was obtained when the D1 scFv cDNA was expressed under a seed-specific Glutelin promoter rather than under the constitutive Ubiquitin promoter. Translational fusion of ER retention signal significantly improved the accumulation of recombinant antibody. Furthermore, lines without ER retention signal lost D1 scFv accumulation in T2 grains. Pilot scale purification was performed for a T2 grain pool (51 g) containing 55.0 mg D1 scFv/kg grains. The crude extract was purified by a two-step purification protocol including IMAC and size exclusion chromatography. The purification resulted in a yield of 0.47 mg of D1 scFv (31 kD) with high purity. Enzyme-linked immunosorbent assay revealed that 29 % of the purified protein was fully functional. In immunoprecipitation assay the purified D1 scFv recognized the native 18 kD BLG in the milk sample. No binding was observed with the heat-treated milk sample, as expected. The developed barley-based expression system clearly demonstrated its potential for application in the processing of dairy milk products as well as in detecting allergens from foods possibly contaminated by bovine milk.
VTT Technical Research Centre of Finland, Tietotie 2, P.O. Box 1000, 02044 VTT, Espoo, Finland, anneli.ritala@vtt.fi.
A
A
Ritala
S
S
Leelavathi
K-M
KM
Oksman-Caldentey
V S
VS
Reddy
M-L
ML
Laukkanen
D000818:Animals; D000917:Antibody Formation; D002417:Cattle; D001467:Hordeum / Q000235:genetics / Q000254:growth & development; D007073:Immunoglobulin E / Q000096:biosynthesis* / Q000235:genetics; D007782:Lactoglobulins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D016269:Milk Hypersensitivity / Q000175:diagnosis / Q000235:genetics / Q000276:immunology; D030821:Plants, Genetically Modified / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics
D007782:Lactoglobulins; D011994:Recombinant Proteins; D007073:Immunoglobulin E