Molecular cloning of recombinant fibronectin EDA and EDB fusion protein.
- Author:
Ai-min XUE
1
;
Hua WANG
;
Rong YE
Author Information
1. Department of Forensic Medicine, Shanghai Medical School, Fudan University, Shanghai 200032. aimin-xue@yahoo.com.cn
- Publication Type:Research Support, Non-U.S. Gov't
- MeSH:
Blotting, Western;
Cloning, Molecular;
Escherichia coli/metabolism*;
Fibronectins/biosynthesis*;
Plasmids;
Polymerase Chain Reaction;
Recombinant Fusion Proteins/isolation & purification*;
Recombinant Proteins/isolation & purification*
- From:
Journal of Forensic Medicine
2002;18(3):140-143
- CountryChina
- Language:Chinese
-
Abstract:
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.
