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Ligand binding by a recombinant insect juvenile hormone binding protein
K Touhara1, K A Lerro, B C Bonning
1Department of Chemistry, State University of New York, Stony Brook 11794-3400.
Biochemistry
|March 2, 1993
Summary
Researchers expressed insect juvenile hormone binding protein (JHBP) in cell lines, creating a recombinant JHBP (rJHBP). This rJHBP binds juvenile hormones with high affinity and is suitable for further structural and functional analysis.
Area of Science:
- Biochemistry
- Molecular Biology
- Insect Physiology
Background:
- Juvenile hormone binding protein (JHBP) plays a crucial role in insect development.
- Understanding JHBP's structure and function is key to insect growth regulation.
Purpose of the Study:
- To isolate, sequence, and express the hemolymph JHBP from larval Manduca sexta.
- To characterize the recombinant JHBP (rJHBP) for its binding affinity and structural properties.
Main Methods:
- cDNA isolation and sequencing of Manduca sexta JHBP.
- Recombinant baculovirus construction for expression in insect cells (Sf9).
- Purification of rJHBP using chromatography and characterization via spectroscopy and ligand binding assays.
Main Results:
- High-level secretion of functional rJHBP (> 50 micrograms/mL) from infected insect cells.
- rJHBP demonstrated higher binding affinity for juvenile hormones (JH I and JH II) compared to native JHBP.
- Circular dichroism spectroscopy revealed secondary structure (34% alpha-helix, 23% beta-sheet) without ligand-induced changes.
Conclusions:
- Recombinant JHBP expressed in insect cells is a functional protein with high affinity for juvenile hormones.
- The expressed rJHBP is suitable for detailed structural and functional studies, aiding in understanding insect hormone regulation.