Comparison of Two Methods in Analysis of Binding Characteristics of Odorant Binding Proteins AlucOBP21 of Apolygus lucorum
[Objective]The objective of this study is to compare two methods of fluorescence competitive binding assay and microscale thermophoresis (MST) in analysis of binding characteristics of odorant binding proteins AlucOBP21 of Apolygus lucorum, and to explore a new method for determination of binding function of insect OBPs. [Method] Total RNA was extracted from antennae of both female and male A. lucorum adults by using Trizol reagent. The cDNAs were synthesized using the Superscript III Reverse Transcriptase system. AlucOBP21 was PCR-amplified using gene-specific primers. The sample cDNA of the antennae was used as the template. The PCR product was cloned into an expression vector PET-32a (+) for expression in prokaryotic BL21 (DE3) cells. The transformation of the strain with pET32a/AlucOBP21 was incubated in cultures and the crude protein with His-tag was obtained. The supernatant was obtained by sonication, purified by Ni ion affinity chromatography. Soon after the His-tag was removed with recombinant enterokinase, then the purified AlucOBP21 without His-tag was harvested. To investigate the binding abilities of AlucOBP21 to candidate ligand chemicals, fluorescence binding test was performed using 1-N-phenylnaphthylamine (1-NPN) as a fluorescence probe. 1-NPN and odor standard samples were dissolved in methanol (mass spectrometry grade). And also, the binding characteristics of recombinant AlucOBP21 to candidate ligands were explored by MST technique. In this assay, the candidate ligand samples were dissolved in dimethyl sulfoxide (DMSO) solution. Candidate ligands included 8 potential sex pheromones and sex pheromone analogues of mirids, 12 green leaf volatiles and a repellent (dimethyl disulfide) of A. lucorum.[Result]Recombinant AlucOBP21 was expressed in both supernatant and inclusion bodies. Finally, the supernatant fraction was selected to purify the target protein. Recombinant AlucOBP21 without His-tag was obtained by using recombinant entherokinase at 22℃. In fluorescence competitive binding assays, recombinant AlucOBP21 could bind with 1-NPN probe, dissociation constant was (6.88±0.31) μmol·L-1. AlucOBP21 could bind with β-ionone and β-caryophyllene, and the dissociation constants were (13.74±1.93) and (13.24±2.12) μmol·L-1, respectively. However, remaining candidate ligands could not effectively bind to recombinant AlucOBP21. Analysis of MST demonstrated that AlucOBP21 could bind with β-ionone, β-caryophyllene, β-pinene and limonene, and the dissociation constants were (0.20±0.02), (0.05±0.01), (0.70±0.04) and (0.40±0.06) μmol·L-1, respectively. The thermophoresis of remaining candidate ligands with AlucOBP21 could not display regular change along with the change of ligand concentration. Therefore, these odorant chemicals could not bind with recombinant AlucOBP21.[Conclusion]In comparison with fluorescence competitive binding assay, MST detection demonstrated a broader ligand spectrum which could not miss odorant ligands with weak binding affinities.
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