Expression Of Mouse Homeobox Gene Pem In E.coli

Background: Members of Homeobox gene family largely encode transcription factors that are required in key developmental events. Mouse homeobox gene Pem was newly isolated in 1990's. Its expression pattern during the murine ontogeny and in the adult tissues is unique, namely, in a stage and spatial specific manner. Although it is tightly regulated in normal tissues, it has been found continuously express in a variety of immortalized and malignant rodent cell lines.To date, extensive research has been reported about the structure and regulation of Pem itself. Nevertheless, little has known about what Pern's bonafide function is. To our knowledge, Pem belongs to Homeobox gene family and may encode a developmental regulated transcription factor. Pem protein may cooperate with other cofactors and by means of which regulates the transcription of subordinate downstream genes. Pem protein preparation is necessary for both anti-sera producing and the in vitro study of its function.Objectives: to express mPem protein in E. coli.Methods: The mouse Pem gene (mPem) cDNA coding sequence was cloned into prokaryotic GST fusion protein expression plasmid pGEX-4T-3. The recombinant plasmid was transformed to E. coli BL21 and the GST/mPem fusion protein was induced to express with IPTG. The fusion protein was purified by affinity chromatography. Then it was subjected to hydroxylamine cleavage to verify the predicted primary structure.Result: GST/mPem fusion protein was expressed successfully in E. coli. We obtained certain amount of GST/mPem protein by means of affinity chromatography. The fusion protein had expected primary structure.Conclusion: pGEX-4T/mPem expression plasmid was constructed. GST/mPem fusionprotein was successfully expressed.