High-efficiency Expression, Purification And Function Study Of The B-cell Receptor-related Membrane Protein BmBCRA Of The Polyhedrin Fusion

Membrane protein is a class of proteins which are contained in biomembranesystems.They are the main excutor for the function of biomembrane.Their Stronghydrophobicity?cell toxicity and other reasons result in great difficulty in expression,purification and crystallization of membrane proteins.Polyhedrin (Polh) can promotethe expression of foreign genes.Inclusion body particles composed by fusionexpression product can dissolved in alkaline buffer. Making use of the properties ofpolyhedra, target proteins can be conveniently purified by regulating pH andcentrifugation.Silkworm baculovirus expression system is an efficient eukaryoticexpression systems.It has high expression and the process of post-translationalmodification.Under the action of strong promoter of polyhedrin,this system canexpress some proteins that are very difficult to express,such as membrane proteins.Bcell receptor-associated protein31(BCRA/BAP31) belongs to B cell receptor-relatedprotein family.It is involved in the process of B cell activation and vesicular transportand has anti-apoptotic function.In silkworm,the related research reports are very few.We screened a gene which encoding B cell receptor-associated protein?BmBCRA?from the cDNA library of Bombyx mori in our laboratory. Its accessionnumber in Genbank is AK386415.1. We constructed a expression vectorpET-32a-Polh-BmBCRA and highly expressed the fusion protein in E.coli BL21(DE3). BmBCRA was not expressed individually,which shows that polyhedrin canpromote the expression of membrane protein.According to the feature of polyhedrin,we purified fusion protein Polh-BmBCR by regulating pH and gel filtrationchromatography and successfully identify it by MALDI-TOF-MS.We also made theantibody of Polh-BmBCR. After digested with TEV protease,we obtained membraneprotein BmBCRA which was removed the Polh tag. We constructed recombinantviruses vPolh-BmBCRA using Bac-to-Bac system and infected BmN cells.Theanalysis of western blotting showed that the fusion protein was successfully expressedin infected cell,while BmBCRA was not expressed individually, which shows that polyhedrin can also promote expression in eukaryotic cells.We also constructedrecombinant virus vEGFP and vBmBCRA-EGFP and respectively infected BmNcells.Infected cells were observed under fluorescence microscope.Comparing with thecells which express EGFP, the green fluorescence of the cells which expressBmBCRA-EGFP is mainly in the vicinity of membrane.The result shows thatBmBCRA is maybe expressed in the form of membrane protein in eukaryoticcells.The result of real-time fluorescent quantificational PCR shows that transcriptionlevel is the highest in malpighian body, followed by the gut and the gonad,lowest inhead and silk gland.Malpighian body is the main excretion organs. BmBCRA proteinsmay plays a significant role in the silkworm excretion system.After RNA interferenceof BmBCRA, the result of detection by MTT and flow cytometry shows that cellvitality is reduced and the rate of apoptosis was significantly increased. This result isconsistent with the anti-apoptotic function of human B-cell receptor-related protein(BAP31).This study mainly attempts to establish a common method to highly expressand purify membrane proteins using polyhedrin fusion tag, which lay a solidfoundation for the crystallization of membrane proteins and functional studies.