The Expression Research of PMP22 Gene in Bombyx Mori
|School||Zhejiang University of Technology|
|Course||Biochemistry and Molecular Biology|
|Keywords||Bombyx mori PMP22 gene membrane protein Bac-to-Bac expression system Western blot|
Membrane proteins plays a very important role in the cell activities, but the expression and preparation of membrane proteins is quite difficult. We fused the polyhedrin protein with the membrane protein PMP22 to explore high level expression methods of this membrane proteins. A gene was screened with an open reading frame (ORF) of 561 bp which encodes 186 amino acids residues form the cDNA library of bombyx mori constructed by our laboratory. Bioinformatics analysis showed that it was the bombyx mori peroxisomal membrane protein whose predicted molecular weight is 22 kD and isoelectric point is 10.41. The protein contains a Mpv17_PMP22 conserved domain superfamily and 4 strong hydrophobic transmembrane region as a integral membrane protein. The gene was named as PMP22 (Peroxisomal membrane protein 22). GenBank accession number is LOC733112.We inserted the ORF of PMP22 gene into the multiple cloning sites of plasmid pBacPAK8-Ph then obtained recombinant gene Ph-PMP22. After that we cut off Ph-PMP22 by double digesting with restriction enzymes and cloned it into the prokaryotic expression vector pET-28a, successfully constructed the recombinant expression plasmid pET-28a-Ph-PMP22. The result of PCR, double diagesting analysis and sequencing identification was proofed that is no mistake. The recombinant plasmids was respectively transformed into E. coli BL21 star (DE3), E. coli BL21 (DE3) and E. coli Rosetta (DE3). We screened the engineering bacterias from the three and cultured them respectively, then induced them by IPTG with a final concentration of 0.5 mmol / L.We disrupt the cells by heating of 100℃and carried a SDS-PAGE electrophoresis. The results showed threre is no significant specific expression even increased the induction time; Western blot detected some expression of fusion proteins in E. coli Rosetta, and SDS-PAGE scan analysis showed the percents of fusion protein in total expression is about 2%.Through Bac-to-Bac system, we recombined the Ph-PMP22 constructed in prokaryotic expression system with cloning plasmid pFastBac to get pFastBac-Ph-PMP22. After acquiring the positive results of identification by PCR, double digesting analysis and sequencing, pFastBac-Ph-PMP22 transformed E. coli DH10Bac with the shuttle vector Bacmid and then transposed with Bacmid to acquire the recombinant Bacmid PCR with pUC/M13 primers proofed the success of the recombination. Bombyx mori BmN cells was infected with the recombinant Bacmid, then was extracted and disrupted for SDS-PAGE electrophoresis showed no obvious specific expression. Western blot analysis suggested that there is no fusion proteins in upper fluid and a small amount of fusion proteins in the precipitation which is lower than prokaryotic expression system.