Studies On Bombyx Mori Major Facilitator Superfamily Protein BmMFS Gene

The membrane proteins are essential for life activities in the organism, mainly responsible forintracellular environment of matter and energy exchange. Because most of the drugs in the targetmembrane proteins, the structural analysis of membrane proteins for drug design is very important.However, low levels of the natural membrane proteins, purification difficulties, shortcomingscloning and expression and other factors led to the slow progress. Major facilitatorsuperfamily(MFS) is a major secondary membrane transporter superfamily. Because of the diversityof substrate transportting, they play an important role in cellular energy metabolism and substanceexchange process. MFS superfamily exists in the lower organisms and higher organisms extensively,is consisted of many members.There are many difficulties in the analysis of membrane proteinsstructure, only five kinds of MFS proteins structure from bacteria is parsed.There is not relatedreports of the protein in silkworm. Polyhedrin(Polh)is a late highly expressed structural protein afterinsect baculovirus infected insects, molecular weight is about29kD, the formation of the inclusionbodies are dissolved in the buffer of pH10.8or above.The N port from amino acid residues20fragment (Ph20) of polyhedrin remains polyhedrin crystal characteristics, and correction of pH to8.0, the fusion protein remained in the supernatant.We use the feature to fuse exogenous proteinswith Ph20to express,then simply purify target protein by adjusting pH.We screened a protein that contains six transmembrane domains in our laboratory silkwormprotein library.It contains a conserved domain of MFS,so we name it as BmMFS. Constructrecombinant plasmid pET-32a-Ph20-BmMFS,and introduce thrombin restriction sites nucleic acidsequences between Ph20and BmMFS gene. Transformed E.coli Rosetta,1mM IPTG induction offusion protein expression.Use the characteristic of Ph20dissolution by adjusting the pH forpreliminary purification, nickel affinity chromatography was further purified, the fusion protein isPh20-BmMFS by Western blotting and mass spectrometry. Using the baculovirus expression systemfor recombinant virus vPh20-BmMFS, infecting BmN cells.When the cell disease,we detect themby western blotting.The detection showed that the fusion protein was successfully expressed.Theresults of Quantitative PCR showed that the transcript levels of BmMFS gene in the organizations ofthe fifth instar larvae of Bombyx mori have significant differences.The highest is the gut,the lowestis the trachea. The analysis of subcellular localization showed that BmMFS exists in the cell membrane.Apply the technology of RNA interference to research function of BmMFS gene atlast,the results showed that the silencing of the gene expression,the rate of cell apoptosisincreased.The fusion expression of Ph20polyhedrin protein fragments and membraneproteins,increased the expression quantity of membrane proteins,simplify the purification process,lay a foundation for the crystallization of membrane proteins.