Dissertation > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)

Fusion Expression and Purification of a New Membrane Protein Gene Named HW from Silkworm

Author ZhangYaNing
Tutor ZhangYaoZhou
School Zhejiang University of Technology
Course Biochemistry and Molecular Biology
Keywords Transmenbrane protein HW gene polyhedrin-fused expression Bac-to-Bac System
Type Master's thesis
Year 2011
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A cDNA sequence was screened from a Bombyx mori cDNA library constructed by our labortary. Its translational product has three trans-membrane areas and is an unknown protein which we named HW. The relevant information about this gene were obtained by Blast against NCBI datebase. Anlysising by bioinformatics we finded that the length of the cDNA of HW gene is 1230 bp and it contains an ORF of 294 bp, encoding 97 amino acid residues with the predicted molecular weight of 10.93 kD.A pair of primers was designed to amplify the coding region of HW gene. The ORF of this gene was cloned into pBacPAK8-Ph vector constructed by our libortary to construct the recombinant vector pBacPAK8-Ph-HW harboring polyhedrin-HW fused gene. There exists a TEV enzyme gene sequence between polyhedrin gene and HW gene, and we can cut off polyhedrin protein in this site to purify HW protein. This fused gene Ph-HW was digested with BamHⅠ/NotⅠand cloned into pET-28a(+) vector.The recombinant plasmid was transformed into E.coli BL21(DE3) and the recombinantion is right by digestion﹑PCR and gene sequencing . Recombinant protein was expressed successfully in E.coli BL21(DE3) after induced by IPTG with the final concentration of 1 mM at 28℃. The analysis of SDS-PAGE showed that the fusion protein His-Ph-HW was expressed highly in E.coli BL21 with a molecular weight of about 40 kD that was consistent with the theory value. This fused protein was purified based on the recombinant protein solubility and by affinity chromatography. HW protein was obtained by TEV digestion.The recombinant fused protein was also expressed in silkworm cell by the Bac-to-Bac Baculovirus Expression System. This fused gene Ph-HW was cloned into pFastBacTMHTb vector within NotⅠand BamHⅠdigestion site. The recombinant plasmid pFastBac-Ph-HW was transformed into E. coli DH10BacTM and generated recombinant bacmid by transposition of the pFastBac-Ph-HW with bacmid. Recombinant bacmid DNA was transfected into the silkworm cell to generate a recombinant baculovirus. The recombinant baculovirus was amplified and infected the BmN cells. The infected cells were collected and SDS-PAGE as well as Western blot were performed to detect the expression of the recombinant plasmid on the translation level. RNAi experiment indicated the importance of HW gene in silkworm cells. We obtained HW protein coarse crystallization at the base of purified fusion protein. All that should lay a good basis for researching the structure and function of this transmenbrane protein.

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