IMMUNOAFFINITY PURIFICATION OF JUVENILE HORMONE-BINDING PROTEIN FROM GALLERIA-MELLONELLA HEMOLYMPH

Previously described methods of purification of hemolymph juvenile hor mone-binding protein (hJHBP) from Lepidoptera were tedious and require d multiple steps. These methods resulted in low protein yield (Kramer et al., 1976; Goodman et al., 1978; Peterson et al., 1982; Park et al. , 1993; Ozyhar & Kochman, 1987). In this report a simple method of pur ification of hJHBP from Galleria mellonella (L.) larvae is described. Monoclonal antibodies against hJHBP were obtained and crosslinked to C NBr-activated Sepharose 4B. The hemolymph of G. mellonella was centrif uged and then chromatographed on Sephadex G-200 gel filtration column. Juvenile-hormone-binding activity containing material from Sephadex G -200 column was subjected to purification on an immunoaffinity column. Bound protein was eluted from anti-hJHBP Sepharose 4B gel by lowering pH to 3.0 with 200 mM citric acid 200 mM Na2HPO4 buffer. This method resulted in 320-fold purification of G. mellonella hJHBP with 56% yiel d.