Bacterial expression and purification of vertebrate odorant-binding proteins

Brulé, Marine; Glaz, Margot; Belloir, Christine; Poirier, Nicolas; Moitrier, Lucie; Neiers, Fabrice; Briand, Loı̈c

doi:10.1016/bs.mie.2020.05.002

Cited by 3 publications

(12 citation statements)

References 32 publications

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“…Recombinant mOBP5 was produced as previously described [42]. Briefly, the cDNA sequence encoding Mus musculus mOBP5 (formerly known as mOBP1a, UniProt ID: Q9D3H2) minus its signal sequence was synthesized using Genewiz (Leipzig, Germany) with codon usage optimized for expression in E. coli.…”

Section: Mobp5 Production and Purificationmentioning

confidence: 99%

“…Circular dichroism (CD) spectra were recorded as previously described [42] using a JASCO J-815 spectropolarimeter (Jasco, Tokyo, Japan) equipped with a Peltier temperaturecontrol system (JASCO MPTC-490S). CD spectra were recorded at 20 • C using a 0.01 cm thick quartz cell between 180 and 250 nm at 0.5 nm intervals with a 50 nm/min scan speed.…”

Section: Recombinant Mobp5 Characterizationmentioning

confidence: 99%

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Ligand Binding Properties of Odorant-Binding Protein OBP5 from Mus musculus

Moitrier

Belloir

Lalis

et al. 2022

Biology

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Odorant-binding proteins (OBPs) are abundant soluble proteins secreted in the nasal mucus of a variety of species that are believed to be involved in the transport of odorants toward olfactory receptors. In this study, we report the functional characterization of mouse OBP5 (mOBP5). mOBP5 was recombinantly expressed as a hexahistidine-tagged protein in bacteria and purified using metal affinity chromatography. The oligomeric state and secondary structure composition of mOBP5 were investigated using gel filtration and circular dichroism spectroscopy. Fluorescent experiments revealed that mOBP5 interacts with the fluorescent probe N-phenyl naphthylamine (NPN) with micromolar affinity. Competitive binding experiments with 40 odorants indicated that mOBP5 binds a restricted number of odorants with good affinity. Isothermal titration calorimetry (ITC) confirmed that mOBP5 binds these compounds with association constants in the low micromolar range. Finally, protein homology modeling and molecular docking analysis indicated the amino acid residues of mOBP5 that determine its binding properties.

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Section: Mobp5 Production and Purificationmentioning

confidence: 99%

Section: Recombinant Mobp5 Characterizationmentioning

confidence: 99%

Ligand Binding Properties of Odorant-Binding Protein OBP5 from Mus musculus

Moitrier

Belloir

Lalis

et al. 2022

Biology

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“…Cells were lysed by sonication yielding the protein in the pellet, which was then dissolved in 8 M urea/5 mM DTT. Solubilised protein was refolded by extensive dialysis against 50 mM Tris-HCl, pH 7.4 and finally purified by anion-exchange chromatography on HiPrep-Q 16/10 20 ml (Bio-Rad, United States), using a linear gradient of 0-1 M sodium chloride in 50 mM Tris-HCl, pH 7.4 (Liu et al, 2014;Brulé et al, 2020).…”

Section: Bacterial Expression and Purification Of Proteinsmentioning

confidence: 99%

Reverse chemical ecology indicates long-chain aldehydes as new potential semiochemicals for the African elephant Loxodonta africana

et al. 2022

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Chemical communication between sexes in the elephants has been well studied at the chemical and behavioural levels, but little is known about the proteins mediating the exchange of chemical signals. Two sex pheromones have been identified in Asian elephants: (Z)-7-dodecenyl acetate and frontalin, and their effects on the elephants’ behaviour have been described in detail. The genomes of both the Asian (Elephas maximus) and the African elephant (Loxodonta africana) have been poorly annotated. In particular, the complete sequences of two odorant-binding proteins and a VEG protein are available for the African elephant, together with isoforms and other members of the same families, which however are incomplete or unreliable. In a previous study, we have expressed the OBP1 of both elephant species, and investigated their binding properties. We showed that OBP1 is tuned to the pheromone (Z)-7-dodecenyl acetate and few structurally related linear esters, but also binds (E)-β-farnesene and farnesol with good affinity. In this work we have explored the characteristics of the second OBP of the African elephant (LafrOBP2). This protein, which was not found in the trunk wash, does not bind any of the above listed semiochemicals. Instead, it shows selected affinity to unsaturated linear aldehydes of 16 carbon atoms, specifically (Z)-9-hexadecenal, (Z)-11-hexadecenal and (10E,12Z)-hexadecadienal (bombykal). Fourteen and 18 carbon orthologues show only much reduced binding affinity. Some linear alcohols, fatty acids and esters also weakly bind this protein with dissociation constants about one order of magnitude higher.

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“… Being the natural carriers for odorant and pheromones, with dissociation constants in the micromolar range, they represent the best candidates to trap and detect environmental odors. OBPs can be expressed in high yields in bacterial and eucaryotic systems and can be easily purified using standard protocols [ 11 ]. Based on the large amount of structural information [ 12 , 13 ], site-specific mutagenesis is easy to design with great confidence of success.…”

Section: Introductionmentioning

confidence: 99%

“…OBPs can be expressed in high yields in bacterial and eucaryotic systems and can be easily purified using standard protocols [ 11 ].…”

Section: Introductionmentioning

confidence: 99%

Responses of the Pheromone-Binding Protein of the Silk Moth Bombyx mori on a Graphene Biosensor Match Binding Constants in Solution

Bonazza

Zhu

Hasler

et al. 2021

Sensors

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An electronic biosensor for odors was assembled by immobilizing the silk moth Bombyx mori pheromone binding protein (BmorPBP1) on a reduced graphene oxide surface of a field-effect transistor. At physiological pH, the sensor detects the B. mori pheromones, bombykol and bombykal, with good affinity and specificity. Among the other odorants tested, only eugenol elicited a strong signal, while terpenoids and other odorants (linalool, geraniol, isoamyl acetate, and 2-isobutyl-3-methoxypyrazine) produced only very weak responses. Parallel binding assays were performed with the same protein and the same ligands, using the common fluorescence approach adopted for similar proteins. The results are in good agreement with the sensor’s responses: bombykol and bombykal, together with eugenol, proved to be strong ligands, while the other compounds showed only poor affinity. When tested at pH 4, the protein failed to bind bombykol both in solution and when immobilized on the sensor. This result further indicates that the BmorPBP1 retains its full activity when immobilized on a surface, including the conformational change observed in acidic conditions. The good agreement between fluorescence assays and sensor responses suggests that ligand-binding assays in solution can be used to screen mutants of a binding protein when selecting the best form to be immobilized on a biosensor.

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Bacterial expression and purification of vertebrate odorant-binding proteins

Cited by 3 publications

References 32 publications

Ligand Binding Properties of Odorant-Binding Protein OBP5 from Mus musculus

Ligand Binding Properties of Odorant-Binding Protein OBP5 from Mus musculus

Reverse chemical ecology indicates long-chain aldehydes as new potential semiochemicals for the African elephant Loxodonta africana

Responses of the Pheromone-Binding Protein of the Silk Moth Bombyx mori on a Graphene Biosensor Match Binding Constants in Solution

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