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Functional analysis of general odorant binding protein 2 from the meadow moth, Loxostege sticticalis L. (Lepidoptera: Pyralidae).

Yin J, Feng H, Sun H, Xi J, Cao Y, Li K - PLoS ONE (2012)

Bottom Line: In this study, the general odorant binding protein 2 gene was cloned from the antennae of Loxostege sticticalis, using reverse transcription PCR and rapid amplification of cDNA ends.Ligand-binding experiments using N-phenyl-naphthylamine (1-NPN) as a fluorescent probe demonstrated that the LstiGOBP2 protein has binding affinity to a broad range of odorants.Most importantly, trans-11-tetradecen-1-yl acetate, the pheromone component of Loxostege sticticalis, and trans-2-hexenal and cis-3-hexen-1-ol, the most abundant plant volatiles in essential oils extracted from host plants, had high binding affinities to LstiGOBP2 and elicited strong electrophysiological responses from the antennae of adults.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China. jyin@ippcaas.cn

ABSTRACT
Odorant binding proteins play a crucial role in transporting semiochemicals across the sensillum lymph to olfactory receptors within the insect antennal sensilla. In this study, the general odorant binding protein 2 gene was cloned from the antennae of Loxostege sticticalis, using reverse transcription PCR and rapid amplification of cDNA ends. Recombinant LstiGOBP2 was expressed in Escherichia coli and purified by Ni ion affinity chromatography. Real-time PCR assays indicated that LstiGOBP2 mRNA is expressed mainly in adult antennae, with expression levels differing with developmental age. Ligand-binding experiments using N-phenyl-naphthylamine (1-NPN) as a fluorescent probe demonstrated that the LstiGOBP2 protein has binding affinity to a broad range of odorants. Most importantly, trans-11-tetradecen-1-yl acetate, the pheromone component of Loxostege sticticalis, and trans-2-hexenal and cis-3-hexen-1-ol, the most abundant plant volatiles in essential oils extracted from host plants, had high binding affinities to LstiGOBP2 and elicited strong electrophysiological responses from the antennae of adults.

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SDS-PAGE electrophoretic and western blot analysis of expressed recombine LstiGOBP2.M. Molecular weight marker; 1. Non-induced E. coli pET30; 2. Induced E. coli pET30; 3. Non-induced E. coli LstiGOBP2; 4. Induced E. coli LstiGOBP2; 5. Western blot; 6. Purified protein cleaved His by rbEK.
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pone-0033589-g002: SDS-PAGE electrophoretic and western blot analysis of expressed recombine LstiGOBP2.M. Molecular weight marker; 1. Non-induced E. coli pET30; 2. Induced E. coli pET30; 3. Non-induced E. coli LstiGOBP2; 4. Induced E. coli LstiGOBP2; 5. Western blot; 6. Purified protein cleaved His by rbEK.

Mentions: Recombinant LstiGOBP2 was expressed in E. coli as a completely soluble protein with high yields (more than 20 mg/L). The His-tag of the recombinant protein was removed by rEK. The protein was purified by two rounds of Ni ion affinity chromatography: the first round was intended to purify the recombinant protein from total protein and the second round was intended to divide the His-tag and the uncleaved His-tagged proteins (Fig. 2). The purified recombinant proteins were then tested for their binding properties and used for the production of polyclonal antibodies.


Functional analysis of general odorant binding protein 2 from the meadow moth, Loxostege sticticalis L. (Lepidoptera: Pyralidae).

Yin J, Feng H, Sun H, Xi J, Cao Y, Li K - PLoS ONE (2012)

SDS-PAGE electrophoretic and western blot analysis of expressed recombine LstiGOBP2.M. Molecular weight marker; 1. Non-induced E. coli pET30; 2. Induced E. coli pET30; 3. Non-induced E. coli LstiGOBP2; 4. Induced E. coli LstiGOBP2; 5. Western blot; 6. Purified protein cleaved His by rbEK.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316592&req=5

pone-0033589-g002: SDS-PAGE electrophoretic and western blot analysis of expressed recombine LstiGOBP2.M. Molecular weight marker; 1. Non-induced E. coli pET30; 2. Induced E. coli pET30; 3. Non-induced E. coli LstiGOBP2; 4. Induced E. coli LstiGOBP2; 5. Western blot; 6. Purified protein cleaved His by rbEK.
Mentions: Recombinant LstiGOBP2 was expressed in E. coli as a completely soluble protein with high yields (more than 20 mg/L). The His-tag of the recombinant protein was removed by rEK. The protein was purified by two rounds of Ni ion affinity chromatography: the first round was intended to purify the recombinant protein from total protein and the second round was intended to divide the His-tag and the uncleaved His-tagged proteins (Fig. 2). The purified recombinant proteins were then tested for their binding properties and used for the production of polyclonal antibodies.

Bottom Line: In this study, the general odorant binding protein 2 gene was cloned from the antennae of Loxostege sticticalis, using reverse transcription PCR and rapid amplification of cDNA ends.Ligand-binding experiments using N-phenyl-naphthylamine (1-NPN) as a fluorescent probe demonstrated that the LstiGOBP2 protein has binding affinity to a broad range of odorants.Most importantly, trans-11-tetradecen-1-yl acetate, the pheromone component of Loxostege sticticalis, and trans-2-hexenal and cis-3-hexen-1-ol, the most abundant plant volatiles in essential oils extracted from host plants, had high binding affinities to LstiGOBP2 and elicited strong electrophysiological responses from the antennae of adults.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China. jyin@ippcaas.cn

ABSTRACT
Odorant binding proteins play a crucial role in transporting semiochemicals across the sensillum lymph to olfactory receptors within the insect antennal sensilla. In this study, the general odorant binding protein 2 gene was cloned from the antennae of Loxostege sticticalis, using reverse transcription PCR and rapid amplification of cDNA ends. Recombinant LstiGOBP2 was expressed in Escherichia coli and purified by Ni ion affinity chromatography. Real-time PCR assays indicated that LstiGOBP2 mRNA is expressed mainly in adult antennae, with expression levels differing with developmental age. Ligand-binding experiments using N-phenyl-naphthylamine (1-NPN) as a fluorescent probe demonstrated that the LstiGOBP2 protein has binding affinity to a broad range of odorants. Most importantly, trans-11-tetradecen-1-yl acetate, the pheromone component of Loxostege sticticalis, and trans-2-hexenal and cis-3-hexen-1-ol, the most abundant plant volatiles in essential oils extracted from host plants, had high binding affinities to LstiGOBP2 and elicited strong electrophysiological responses from the antennae of adults.

Show MeSH
Related in: MedlinePlus