Biochemical characterization and bacterial expression of an odorant-binding protein from Locusta migratoria.
Analysis of soluble proteins from different body parts of Locusta migratoria revealed a fast-migrating component in native electrophoresis, unique to antennae of both sexes. N-terminal sequence analysis and cloning identified this protein as a member of the insect odorant-binding proteins, carrying a well-conserved six-cysteine motif. Mass spectrometry analysis confirmed the occurrence of two distinct polypeptide species determined by nucleotide sequencing and demonstrated that the cysteine residues are paired in an interlocked fashion. The protein was expressed in a bacterial system with yields of about 10 mg/l of culture, mostly present as inclusion bodies. However, this recombinant product was solubilized after disulfide reduction. Air oxidation yielded a species with all disulfides spontaneously formed as in the native counterpart. Both native and recombinant proteins migrated as a dimer in gel filtration chromatography. Ligand binding was measured, using N-phenyl-1-naphthylamine as the fluorescent probe; the affinity of other ligands was measured in competitive binding assays. The protein exhibited great resistance to thermal denaturation even following prolonged treatment at 100 degrees C. A structural model for this dimeric species was generated on the basis of its sequence homology with Bombyx mori pheromone-binding protein, whose three-dimensional structure has been resolved as an unbound species and in complex with its physiological ligand. This is the first report of an odorant-binding protein identified and characterized from Orthoptera.
Ban
L
L
Department of Entomology, China Agricultural University, Beijing, China.
Scaloni
A
A
D'Ambrosio
C
C
Zhang
L
L
Yahn
Y
Y
Pelosi
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D019281:Dimerization; D004220:Disulfides / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006358:Hot Temperature; D002479:Inclusion Bodies / Q000378:metabolism; D019476:Insect Proteins / Q000737:chemistry* / Q000378:metabolism*; D008024:Ligands; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009987:Orthoptera / Q000737:chemistry*; D011485:Protein Binding; D018035:Receptors, Odorant / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D004220:Disulfides; D019476:Insect Proteins; D008024:Ligands; D018035:Receptors, Odorant; D011994:Recombinant Proteins; C045648:odorant-binding protein