Expression And Functional Analysis Of Emc Protein From Silkworm (Bombyx Mori)

extramacrochaetae (emc) is a member of the Helix-Loop-Helix (HLH) superfamily. It lacks the basic region thus is unable to bind DNA and is a negative regulator of other bHLH proteins by forming inactive heterodimers with them. The Bmemc cDNA was 1619 bp in length, containing a 429 bp ORF, which encode a polypeptide of 142 amino acids; the molecular weight of this protein was estimated to be 15.59 kD. We obtained the ORF of the Bmemc from the total cDNA of silkworm pupa by PCR. After being digested with EcoR I and Hind III, the DNA fragment was inserted into the fusion expression vector pET-28a. The results of PCR and digestion showed that the target fragment was inserted correctly. The recombinant plasmid was transformed into E.coli BL21 (DE3). After being induced by IPTG, the recombinant protein was examined by SDS-PAGE. The most recombinant protein was insoluble inclusion body. In order to obtain abundant soluble recombinant protein, the insoluble inclusion body was solubilized with 4 M urea. The recombinant protein was purified with Ni-affinity chromatography. The molecular weight of the recombinant protein was characterized by mass-spectrum after being purified with 10 RI FPLC. The results indicated that the molecular of the recombinant was 19.28 kD (His tag was weight of 3.69 kD, and Bmemc was weight of 15.59 kD). The purified recombinant protein was used to immunize a male New Zealand rabbit. We have detected the expression of Bmemc in mRNA and protein levels. The results showed that the expression level was highest in the tissues of testis, followed by the ovary, head, midgut and trachea; the protein was barely detectable in the Malpighian tuble, silk gland and fat body. Bmemc is highly expressed in moth, fifth instar larvae and pupa, but lower in egg. Bombyx mori cell line Bm5 was cultured in TC-100 supplemented with 10% fetal bovine serum at 27℃. We blocked the Notch signaling pathway withγ-secretase inhibitor in order to examination the relationship between Bmemc and Notch signaling pathway. The results showed that theγ-secretase inhibitors could reduce the expression level of Bmemc in Bm5 cell. We proposed that Bmemc was regulated by Notch signaling pathway as a target gene.