[Gene cloning, expression and purification of fusion protein epidermal growth factor-linker-trichosanthin].
OBJECTIVE
To construct a recombinant expression vector of the fusion protein epidermal growth factor (EGF)-Linker-trichosanthin (TCS) and achieve its expression in E. coli to obtain purified EGF-linker-TCS fusion protein.


METHODS
The gene fragments of EGF-linker were amplified by PCR and inserted into the expression plasmid PQE30-TCS, followed by transformation of the recombinant plasmid into E. coli M15 for expression of the fusion protein. Ni-FF column chromatography was utilized for purification of the expressed product.


RESULTS
The recombinant plasmid PQE30-EGF-linker-TCS was stably and highly expressed in E. coli M15. The expressed product existed in the form of soluble protein accounting for about 40% of total cellular protein and reached a purity of above 95% after purification with Ni-FF column chromatography.


CONCLUSIONS
The recombinant plasmid PQE30/EGF-linker-TCS has been successfully constructed, which provides a basis for further structural and functional study of EGF and TCS and their potential clinical application for cancer therapy.
Li
Yong-Mei
YM
Department of Traditional Chinese Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. LMX2007@163.com
Yang
Hai-Wen
HW
Luo
Ren
R
D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015978:Trichosanthin / Q000235:genetics* / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D015978:Trichosanthin; D004815:Epidermal Growth Factor