Recombinant Expression And Role In Cell Proliferation Of Human Inhibitor Of Differentiation 3

Id (inhibitors of differentiation/DNA binding) proteins, which inhibit cell differentiation and promote cell proliferation, are negative regulators of basic helix- loop- helix (bHLH) type transcription factor. There are four related members of the Id family called Id1, Id2, Id3 and Id4 in mammalian cells. Id3 was originally identified as a serum-inducible immediate early gene in an established murine fibroblastic cell line. It involved in various biological processes including T and B cell development,skeletal muscle differentiation,vascular smooth muscle cell proliferation,embryonic neurogenesis,osteogenesis and tumor–induced angiogenesis.Human Id3 consists of 119 amino acids with a molecular mass of approximately 13 kDa. In our previous study, the full-length (360bp) Id3 coding region was obtained from human prostatic carcinoma cell line PC-3M by RT-PCR and cloned into pGEM-T. A pair of new primers with EcoR I , Sal I enzymatic digestive sites were designed to amplify Id3 gene by PCR. The recombinant plasmid Id3/pGEM-T was confirmed by enzymatic digestion, PCR reaction and DNA sequencing. The gene sequence inserted in the recombinant vector was totally identical to the published Id3 gene sequence in GeneBank.The recombinant vector Id3/pGEM-T was digested with EcoR I , Sal I and cloned into pET-32a to yield Id3/pET32a plasmid. In Id3/pET32a expression vector, the Id3 frafment was between the two His6 tag coding sequences and under the control of T7 promoter. The Id3/pET32a was transformed into E. coli BL21(DE3) and expressed target protein was induced by IPTG. Contrast to the empty host bacterical lysis, the recombinant plasmid transformed E. coli BL21 expressed a great quantity of fusion protein detected by Western blot. The recombinant plasmid was induced with different induction time, temperature and IPTG concentration. The optimal expression condition for the production of soluble Id3 was perfomed at 25℃, with 0.2 mM IPTG and 8 h afterinduction.The fusion protein was purified with Ni-chelated affinity chromatography. The purity of obtained Id3 was more than 90%. The purity and specificity of purified protein was validated by SDS-PAGE and western blot.To explore the role of Id3 in the proliferation and apoptosis of Daudi cells stimulated with Cisplatin, Daudi cells were treated with Cisplatin in different concentration for 24 h in vitro. The inhibitory effect of cisplatin on Daudi cell proliferation was assayed with Trypan blue staining method. The cell cycle progression and apoptotic rate were determined by the flow cytometry(FCM).Expression of Id3 mRNA of the cisplatin-treated Daudi cell was detected by RT-PCR technique. The resoults revealed that Cisplatin obviously inhibited the Daudi cell growth with dose-dependent manner. After stimulating Daudi cells with Cisplatin at the concentration of 2μg/ml for 24h, cell cycle of Daudi cells was apparently arrested at G1 phase. The proportion of cells in S and G2/M phases were decreased. The apoptotic rate of Cisplatin group (7.04±0.41)% was obviously enhanced in comparison with the control group(4.54±0.67)%(P<0.05). Futhermore, the expression level of Id3 mRNA was significantly higher in Cisplatin group than in control group. Our results suggest that Id3 gene may play an important role in the regulation of cell proliferation and apoptosis of Daudi stimulated with Cisplatin.