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Updated: Aug 12, 2026
Identification of Protein Interacting Partners Using Tandem Affinity Purification
Published on: February 25, 2012
[Eukaryotic expression, purification and analysis of the fusion protein of Epo-Tpo(C)]
1State Key Laboratory for Molecular Virology and Genetic Engineering, Chinese Academy of Preventive Medicine, Beijing 100052, China.
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.
Conclusion:
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.
