[Construction of different mutants of HA-tagged human RAGE gene and their eukaryotic expression].
OBJECTIVE
To construct eukaryotic expression vectors for HA-tagged receptor for advanced glycation end products (RAGE) mutants.


METHODS
Site-directed mutagenesis was applied to wild-type RAGE gene cloned in the pcDNA3 vector with HA tag to obtain the mutants pcDNA3-HA-RAGE(S391A), pcDNA3-HA-RAGE(S399A), pcDNA3-HA-RAGE(S400A), and pcDNA3-HA-RAGE(T401A). After identification by sequencing, the mutants were transfected into HEK293 cells, and the expression of these mutants were detected by Western blotting using anti-HA antibody.


RESULTS
The HA-tagged RAGE mutants constructed were verified successfully by sequencing, and highly expressed in HEK293 cells.


CONCLUSIONS
The success in constructing HA-tagged RAGE mutants, which are highly expressed in eukaryotic cells, may facilitate the functional study of RAGE in cell signal transduction.
Cheng
Wei-wei
WW
Department of Pathophysiology, Southern Medical University, Guangzhou 510515, China. chengweiwei1984@163.com
Li
Yu-sheng
YS
Gong
Xiao-wei
XW
Zhao
Lin-lin
LL
Wang
Ji-gang
JG
Deng
Peng
P
Jiang
Yong
Y
D002460:Cell Line; D003001:Cloning, Molecular; D005057:Eukaryotic Cells / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000096:biosynthesis / Q000235:genetics*
D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic