Open AccessImmunoaffinity purification of juvenile hormone-binding protein from Galleria mellonella hemolymph.
Author(s) -
Elżbieta Wieczorek,
Jan M. Rodriguez Parkitna,
J Szkudlarek,
Andrzej Ożyhar,
Marian Kochman
Publication year - 1996
Publication title -
acta biochimica polonica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.452
H-Index - 78
eISSN - 1734-154X
pISSN - 0001-527X
DOI - 10.18388/abp.1996_4456
Subject(s) - hemolymph , galleria mellonella , sephadex , chromatography , sepharose , size exclusion chromatography , affinity chromatography , juvenile hormone , chemistry , biochemistry , biology , enzyme , hormone , virulence , gene
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.