Prokaryotic Expression, Subcelluar Localization And Functional Analysis Of BmE(spl)-like Gene In Bombyx Mori

We screened a novel gene with an open reading frame (ORF) of 606 bp and encoding 201 amino acidswith a predicted molecular weight of 22.3 kD, isoelectric point was 9.70 from the silkworm pupae cDNAlibrary constructed by our laboratory. After blasting the sequence homology in the NCBI database, weidentified it was a novel gene in the Bombyx mori.(GenBank accession number is GU238288)The proteinpresented here exhibit a high degree of sequence similarity to proteins encoded by E(spl). E(spl) was found toencode highly conserved helix-loop-helix (HLH) proteins which contained the bHLH motif, orange domain,and tryptophan-arginine-proline-tryptophan (W-R-P-W) domain. We named it BmE(spl)-like (Bombyx moriEnhancer of Split like) gene.The ORF of the gene was cloned into the prokaryotic expression vector pET-28a and the recombinantplasmid was transformed into E.coli BL21(DE3). After induced with IPTG, this gene was successfullyexpressed in insoluble inclusion body. The analysis of SDS-PAGE showed that there was a specific proteinstrap. We dissolved the insoluble inclusion body by the solution which contained 8mol/L ureal, and then thefusion protein was purified by affinity chromatograph and 10 RI FPLC. The mass-spectrum analysis indicatedthat the real molecular weight of the fusion protein was 26.28 kD, which was concordance with predicted. Thepurified fusion protein was used to immunize a male New Zealand white rabbit and then the antiserum waspurified by protein A gel column. The titer of the antiserum can arrive to 1:25 600 with a high specificityidentified by ELISA and Western blotting. We extracted the total protein and RNA from different tissues anddevelopmental stages of Bombyx mori, and found that the levels of transcription and expression were greatdifference in the different tissues and developmental stages of Bombyx mori by Western blotting and real-timePCR. Meanwhile, we located the protein in the BmN cells and the result indicated that it was distributed in thecytoplasm. The fusion protein (His-BmE(spl)-like) was used to investigate the binding protein ofBmE(spl)-like by His pull-down. Electrophoresis results showed that there were at least two BmE(spl)-likebinding components, and which molecular weight of were at approximately 45 kD, within each bodycompartment.