Function Study Of Bombyx Mori Nuclear Polyhedrosis Virus Protein P10

Being researched for at least three decades, the specific function of baculovirus protein P10is yet a mystery. It is generally accepted that P10is an abundant viral protein in baculovirus-infected cells and is found associated with fibrillar structures in the cytoplasm and nucleus of thesecells. Other functions of P10such as involvement in polyhedron envelope morphogenesis or therelease of virons from infected cells are far from fully confirmed or understood. To systematicallystudy the function of BmNPV P10, we first did a series of bioinformatics analysis of P10, then wewe developed a subcellular localization predictor to deal with the low specificity of presentpredictors in three steps: first, construction of a dataset intended for Bombyx mori proteins; second,proposal of a feature extracting method that integrates the information of sequence-segmentedamino acid composition and amino acid position; third, classifier training with the support vectormachine algorithm and performance assessment by Jackknife test. The overall accuracy of thepredictor is80.6%. Then the predictor was employed to predict the subcellular location of BmNPVP10in its host cells, result of which indicated nuclei of host cells. The location predicted wasconsistent with that showed by indirect immunofluorescence.In order to reveal the function and its mechanism of P10, it is necessary to get the tertiarystructure of it. The molecular weight of the BmNPV P10protein is only about10kDa, which isnot easy to directly obtain the crystal of its monomers. In this article, we attempted to link BmNPVP10with a carrier protein whose tertiary structure is known and that can be readily crystallizedand explored the expression and purification of fusion proteins. First, given that P10linked to thecarboxyl terminal of manganese superoxide dismutase (MnSOD) is prone to get degraded fromthe P10part of the fusion protein, we constructed a recombinant expression vector pET-28a-p10-MnSOD and expressed it in E. coli. When assayed with polyacrylamide gel electrophoresis (SDS-PAGE) and MS/MS, it was found that the fusion protein is successfully expressed and is partlysoluble in phosphate buffer (PBS, pH7.4). In addition, in view of previous success incrystallization using glutathione S-transferase (GST) fusion protein, this paper also expressed thefusion protein of GST-P10and explored the purification conditions for it. The purified GST-P10fusion protein solution was assayed by SDS-PAGE and result showed that there are two bandswith a7kDa diffirence in molecular weight. After tandem mass spectrometry (MS/MS) identification, both proteins in the two bands are identified as GST-P10.This article developed an appropriate prediction algorithm intended for the silkworm cellprotein to predict the subcellular localization of proteins, offering a novel method for thesubcellular localization prediction of proteins from both Bombyx mori and Bombyx morinucleopolyhedrosis virus. And the subcellular localization of BmNPV P10is validated on theexperimental level; this article also explores the fusion expression and purification of BmNPV P10linked with MnSOD and GST, providing references for the tertiary structure solving and functionanalysis of protein P10.