Prokaryotic expression and purification of fusion protein PTDª²Cyclin D1

    ZHU Yangª²Ting, ZOU Lian, LIANG Yingª²Min, HAN Hua1£¬

    1Department of Medical Genetics and Developmental Biology, School of Basic Medicine, Fourth Military Medical University, Xi¡¯an 710033, China, 2Department of Urology, Second Artillery General Hospital, Beijing 100088, China, 3Center of Stem Cell Medicine,Tangdu Hospital, Fourth Military Medical University, Xi¡¯an 710038, China

    ¡¾Abstract¡¿ AIM: To construct the recombinant expression vector and to obtain large amount of mouse Cyclin D1 protein fused to PTD. METHODS: A 888 bp of mouse Cyclin D1 gene fragment was amplified by PCR method and cloned into pET16b vector immediately downstream of the PTD fragment, an E.coli expression vector, to construct a recombinant plasmid pET16bª²PTDª²CCND1. The plasmid was transformed into E.coli BLª²21 (DE3) and induced to express fusion protein PTDª²Cyclin D1 with IPTG. The expression of PTDª²Cyclin D1 was detected by SDSª²PAGE electrophoresis and Western blot. RESULTS: A novel protein with expected molecular mass was expressed upon induction with IPTG. The expressed product showed good reactivity to antiª²His tag antibody, and was mostly in the form of inclusion bodies. CONCLUSION: Our successful cloning and expression of Cyclin D1 gene and purification of Cyclin D1 protein lay a basis for further study on the application of this protein to accelerate cell proliferation in vitro and in vivo. ......