Immunoaffinity purification of juvenile hormone-binding protein from Galleria mellonella hemolymph.
Previously described methods of purification of hemolymph juvenile hormone-binding protein (hJHBP) from Lepidoptera were tedious and required 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 purification of hJHBP from Galleria mellonella (L.) larvae is described. Monoclonal antibodies against hJHBP were obtained and crosslinked to CNBr-activated Sepharose 4B. The hemolymph of G. mellonella was centrifuged 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% yield.
Wieczorek
E
E
Division of Biochemistry, Technical University of Wrocław, Poland.
Parkitna
J M
JM
Szkudlarek
J
J
Ozyhar
A
A
Kochman
M
M
D000818:Animals; D002352:Carrier Proteins / Q000097:blood / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006458:Hemolymph / Q000378:metabolism*; D019476:Insect Proteins; D007605:Juvenile Hormones / Q000097:blood*; D007915:Lepidoptera
D002352:Carrier Proteins; D019476:Insect Proteins; D007605:Juvenile Hormones; C035606:juvenile hormone-binding protein, insect