[Expression and identification of the extracellular domain of human epidermal growth factor receptor type III variant].
OBJECTIVE
To clone the extracellular domain (ECD) of the type III variant of human epidermal growth factor receptor (EGFRvIII) and construct the recombinant expression plasmid.


METHODS
A DNA fragment (vIII ECD) encoding the extracellular domain of human EGFRvIII was obtained by PCR, and its T-A was cloned and sequenced. The DNA fragment was then ligated into the GST fusion expression vector to construct the recombination plasmid. After identification with restriction digestion and DNA sequencing, the recombinant plasmid was transformed into E. coli BL21 (DE3) for expression of the recombinant protein. The target protein was identified by SDS-PAGE and Western blotting.


RESULTS
The results of restriction digestion and DNA sequencing confirmed the successful construction of the recombinant plasmid. SDS-PAGE showed that the fusion protein was expressed as inclusion bodies in E. coli BL21 (DE3), and the amount of the fusion protein expressed in the bacteria, after induction for 4 h, accounted for up to 15% of the total bacterial proteins. Western blotting demonstrated that the fusion protein could be recognized by the specific anti-EGFR antibody.


CONCLUSIONS
We have successfully constructed the recombinant expression vector of vIII ECD and induced the expression of the fusion protein, which may facilitate functional and immunological studies of EGFRvIII.
Zhang
Xing-mei
XM
Department of Neurobiology, Southern Medical University, Guangzhou 510515, China. zhangxm@smu.edu.cn
Shi
Yu-sheng
YS
Liu
Zhong-ying
ZY
D003001:Cloning, Molecular; D066246:ErbB Receptors / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
D011993:Recombinant Fusion Proteins; C410723:epidermal growth factor receptor VIII; D066246:ErbB Receptors