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[Molecular cloning of recombinant fibronectin EDA and EDB fusion protein]
Ai-min Xue1, Hua Wang, Rong Ye
1Department of Forensic Medicine, Shanghai Medical School, Fudan University, Shanghai 200032. aimin-xue@yahoo.com.cn
Objective:
Construct a recombinant plasmid pET28a-EDA-EDB, prepare the fusion EDA-EDB protein.
Methods:
For the production of recombinant fibronectin EDA-EDB in Escherichia coli, the EDA and EDB segments were separated from pGEM2-EDA/EDB and recomposed with two additional amino acids, then cloned into the expression vector pET28a. pET system to express EDA-EDB fusion protein and 6 x His/Ni-NTA system to purify it in a single step were used. Western blotting confirmed the purified protein.
Results:
The EDA and EDB segments were ligated and inserted into pET28a vector. EDA-EDB fusion protein was highly expressed in Escherichia coli BL21 (DE3). Afterwards, it was purified by Ni-NTA resin and verified by western blotting.
Conclusion:
EDA-EDB fusion protein can be expressed in pET system and purified by 6 x His/Ni-NTA system.