Construction Of Prokaryotic Expression Vector Of A Novel Human Gene Ac3-33and Expression Of GST-fusion Protein

Activator protein-1(AP-1) is an important transcription factor, and the down regulation of its activity has been associated with many human diseases such as tumors. Using luciferase reporter gene screening, we found ac3-33(GenBank name:C3orf33, Accession No. FLJ31139) gene could inhibit the transcriptional activity of AP-1which induced by PMA+Ionomycin. ac3-33is an important novel gene which is widely expressed in various tissues, and AC3-33protein is expressed in cytoplasm, significantly low expressed in human leukemia cell line K562and KG la, it could play an important role in the regulation of AP-1. Further sudies in its molecular mechanisms may provide clues for its potential applications in human disease treatment and diagnosis. In this paper we cloned and constructed of prokaryotic expression vector of ac3-33, expressed and purified of GST-AC3-33fusion protein. This study will establish the foundation for further researches of the function of ac3-33.Using a pair primer containing two restriction enzyme sites of XhoI and BamH I as special primers, ac3-33fragment was amplified from pcDB-TOPO-ac3-33, purified and recycled, then cloned into pGEX-4T-1vector, transformed into DH5a competent cell. After cultured on LB solid medium with ampicillin for16hours, monoclone was selected, and then extracted, purificated and recycled, the plasmid was identified by PCR, restriction enzyme cutting and sequencing.AC3-33fusion protein was expressed in soluble protein and inclusion body in DH5a, the soluble GST-AC3-33fusion protein was focused on this paper. Different kinds of mediums, concentration of NaCl and IPTG, pH, induction temperature and culture time were studied. These results indicated that the optimal conditions for highly expression of soluble protein were:the recombinant trains were cultured in LB medium with100μg/L ampicillin and0.2%NaCl, pH7.0for5-6h at30℃and induced with0.5mM IPTG.The effects of cell-break methods and the concentration of GSH washing solution on the purification of GST-AC3-33fusion protein were studied. The results showed that repeated freezing and thawing, enzymatic hydrolysis and sonioation could break cell and release proteins, and then High-Affinity Resin was used to purify the fusion protein with lOmM reduced glutathione(GSH)elution solution, the concentration of AC3-33fusion protein could reach the level of0.32mg/mL.