[Eukaryotic expression, purification and analysis of the fusion protein of Epo-Tpo(C)].
OBJECTIVE
To further elucidate the function of Tpo(C) and create a new type of Epo.


METHODS
Tpo(C) was linked to Epo to produce a fusion protein--Epo-Tpo(C). The fusion gene was expressed in CHO cell. After selection with G418, Epo-Tpo(C)-expressing cell lines was obtained. The supernatant had 15 U/ml Epo activity. The results of PCR and Southern Blotting indicated the fusion gene was fused into the genome. RT-PCR and RNA Dot Blotting analysis found the Epo-Tpo(C) mRNA. The supernatant was purified with Blue-Sepharose CL-6B.


RESULTS
The rate of recovery was 94%. The in vivo activity tested by reticulocytes was 20% higher than ELISA. The half-life was about 12 hours while the Epo standard sample had half-life of 8 hours tested with ELISA in rat.


CONCLUSIONS
The results proved that Tpo(C) could protect Epo and improve its function. The fusion protein had satisfactory effect in the model of rat renal failure.
Wang
Z
Z
State Key Laboratory for Molecular Virology and Genetic Engineering, Chinese Academy of Preventive Medicine, Beijing 100052, China.
Hou
Y
Y
Yang
X
X
D000818:Animals; D016466:CHO Cells / Q000378:metabolism; D006224:Cricetinae; D004921:Erythropoietin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D013926:Thrombopoietin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D004921:Erythropoietin; D013926:Thrombopoietin