Isolation, Purification And Preliminary Crystallization Research Of BmOBP2in Bombyx Mori

Specific, sensitive olfactory system of insects possess considerable significances in theprogress of foraging and courtship． The recognition of odorant substance in insect is anextraordinarily complex chain-reaction progress, and involves many proteins, such asodorant-binding proteins, odorant receptors, chemosensory proteins and odorant degradingenzymes etc. Odorant-binding proteins (OBPs) are small, water-soluble proteins uniquelyexpressed in olfactory tissue of insects and vertebrates. They may represent the initialbiochemical recognition step in olfaction, because they carrying odorant molecules to theolfactory receptors. It is central to their interaction with their environment and to communicationbetween individuals. The silkworm was considered as a model system for studyingchemosensory communication for the sufficiently sensitive olfactory in silk moth.In this article, by homogenate, differential centrifuge, density gradient centrifuge, highpressure crush and high speed centrifuge. The high quality membrane was obtained, and thenwas extracted with detergent to dissolve these membrane proteins. The solved membraneproteins were further isolated with ion-exchange chromatograph and size exclusionchromatograph using the AKTA explorer10protein purified system. We analysed this proteinwith2D electrophoresis, digging the sepecial protein dots for mass spectrometric analysis. Wegot a hit of this membrane protein in the Southwest University Protein Data Bank is BmOBP2.Meanwhile, the gene coded BmOBP2protein was successfully coloned from silkwormcDNA library, constructed expression vector pET-32a-BmOBP2、pGEX-6P-1-BmOBP2andfusion expression vector pET-28a-MnSOD-BmOBP2successfully. The relative pure protein wasobtained by affinity chromatography and ion-exchange chromatograph for the exploration ofcrystallization conditions. The result of tissue localization showed that the native BmOBP2protein was detected in the head of silkworm puma, but not detected in the body wall and tail.Furthermore, the interaction between BmOBP2and total pupa protein power was investigated byGST pull down technology，mass spectrometry analysis showed that it could be Bmb021422.The study of odorant-binding proteins in Boybmx mori promoted to revealing themechanism of odor perception in insects, and it could be refered to the study of vertebrateolfactory.