High-level Expression And Purification Of Fusion Protein Polh-N141

Membrane protein provide the main function of biomembrane.Study structure and function of membrane protein help us to know what role of it in biomembrane.First,we must purify it.Actually,the membrane protein is difficult to purify,because generally the protein integrate into membrane and a little expression.The thesis studies on making use of polyhedrin to express the fusion protein Polh-N141 highly and purify it in E.coli expression system and Bombyx mori baculovirus expression system.A cDNA sequence was screened from a Bombyx mori membrane protein cDNA library constructed by our labortary,which has a conserved domain chitin_bind₄ identified after Blast based on NCBI database.We preliminary named this novel gene as N141.It contains an ORF of 435 bp and encodes 144 amino acids.It was predicted the molecular weight of 16.9 kDa and has one transmembrane domain.According to the ORF of N141,two primers were designed to obtain the coding region of N141 gene.The ORF of the gene was cloned into pBacPAK8-Polh vector.The first recombinant plasmid pBacPAK8-Polh-N141 was constructed.The fragment of Polh-N141 gene was cloned into pET-28a vector with the first recombinant plasmid by BamH I and EcoRⅠdigest.The second recombinant plasmid pET-28a-Polh-N141 was constructed.Fusion protein was expressed successfully in bacteria containing recombinant plasmid pET-28a-Polh-N141 induced by IPTG.The analysis of SDS-PAGE and Western blotting showed that the fusion protein Polh-N141 was expressed highly and formed into inclusion bodies in deposition.With the strong promoter,fusion protein was expressed highly.The deposition was washed by Tris·Cl buffer of pH 8.0,pH 9.0,pH 10.0,pH 11.0,pH 12.0,which method was based on the property of polyhedrin solubility.The method of purification the fusion protein was named gradient pH of Tris·Cl buffer washing method.The fusion protein with 6-His tag was detected by Western blotting and PMF.The fusion protein was digested by TEV protease to remove polyhedrin,so we can get the cuticular memebrane protein N141.By the property of polyhedrin,the membrane protein was expressed highly and purified easily.Moreover, with Bac-to-Bac baculovirus expression system,the fragment of Polh-N141 gene was cloned into pFastBac HTb vector.The third recombinant plasmid pFastBac-Polh-N141 was constructed and transformed into DH10Bac E.coli which has the transposition with Bacmid.So we can get recombinant Bacmid and transfected into silkworm cell BmN to get recombinant baculovirus vPolh-N141.vPolh-N141 was transfected into silkworm cell BmN again.The analysis of Western blotting showed that fusion protein was expressed in the infected cell.The study was tried to increase the expression of membrane protein and purify it,which was laid the foundation of further research it on the structure and function.