Purification, Bacterial Expression And Biochemical Characterization Of Chemosensory Proteins From Locust

The purification of odorant-binding proteins (OBPs) from Locusta migratoria and chemosensory proteins (CSPs) from L. migratoria and Schistocerca gregaria was performed. The general properties of proteins were characterized and both an OBP and three CSPs have been expressed in the bacterial system with high yield.Fifteen new sequences of CSPs have been identified on the basis of the molecular cloning of cDNAs from the antennae of L. migratoria. The CSPs had calculated molecular weights of 12.2 ~ 12.9kDa, and pI values of 5.1- 6.2. There were a number of isoforms, which could be classified into two sublasses (CSPI and CSPII). All CSPs in L. migratoria had 4 conserved cysteines linked by disulphide bridges between adjacent residues. One of CSP isoforms (CSP/m-II-10) was expressed in a bacterial system (E. coli) with high yield.Several CSP isoforms from antennal, tarsal and wing extracts of L. migratoria were purified and biochemically characterized. For the 15 deduced sequences, there was good agreement between measured and calculated masses only in a few cases.In wings, the main endogenous ligand binding to CSPs was determined as oleoamide by a gas chromatography- mass spectrometric approach.Analysis of soluble proteins from L. migratoria revealed a fast-migrating component specifically located in antennae of both sexes. N-terminal sequence analyzing and cloning identified this protein as a member of the insect OBPs, named Lmig-OBP, which contained two isoforms differing by the single amono acid substitution Asnl9/His19. This is the: first report of an OBP identified and characterized from Orthoptera.Lmig-OBP carried a well-conserved six cysteine motif. Mass spectrometry confirmed the occurrence of two distinct polypeptide species determined by nucleotide sequencing and demonstrated that the cysteine residues were paired in an interlocked fashion. Both native and recombinant proteins migrated together as a dimer in gel filtration chromatography. A structural model for this dimer was obtained from its sequence homology with Bombyx mori pheromone-binding protein.Lmig-OBP was expressed in a bacterial system with yields of about 10 mg/1 of culture, mostly present as inclusion bodies, and was solubilized after disulfide reduction.Polyclonal antibodies to CSPIII expressed in S. gregaria stained a band at 14 kDa in the extracts of antennae and wings of the adults in 5. gregaria, but not in the nymphs. By contrast, tests with the same antibodies in L. migratoria demonstrated cross reaction with a band of a different weight (around 35 kDa) that was found only in the extract from nyimphs, and not from adults.The expression of the OBP, CSPI and CSPII in L. migratoria was also monitored in different organs of both solitary and gregarious locusts in their nymphal and adult stages. OBP was found only in antennae, in both solitary and gregarious locusts, while CSP II was found in several organs and CSPI was common in most organs tested.The recombinant Lmig-OBP and the CSPs (including CSP-sg4 and CSPIII of 5. gregaria and CSP/m-II-10 from L. migratoria) bind reversibly with N-phenyl-1-naphthylamine (1-NPN) in fluorescent-binding assays, with dissociation constants of 1.7, 4.0, 8.0 and 6.2uM.The protein exhibited great resistance to thermal denaturation even following prolonged heating at 100C. Since the probe 1-NPN is the strongest ligand compared with others, it was used as a fluorescent reporter to measure the affinity of other ligands in competitive binding assays. The competitive binding experiments indicated that 2-amylcinnamaldehyde was one of the most competitive ligands, because of structural similarity to 1-NPN.