[Vector construction for the intracellular domain of human receptor for advanced glycation end- products and expression of the fusion protein].
OBJECTIVE
To construct a eukaryotic expression vector for GST-tagged intracellular domain of human receptor for advanced glycation end-products (hRAGE), and to study the function of the expressed fusion protein and identify its interacting proteins.


METHODS
The coding sequence of the intracellular fragment of hRAGE was amplified by PCR and inserted into pGEX-KG vector, a general GST fusion protein expression vector. After PCR identification, endonuclease digestion and DNA sequencing, the recombinant was transformed into E.coli BL21 to achieve the expression of GST fusion protein induced by isopropyl-beta-D-thiogalactoside (IPTG), followed by purification of the protein on glutathione-superflow resin.


RESULTS
The recombinant of GST/hRAGE-C was constructed and identified by PCR, endonucleases digestion and DNA sequencing. After protein expression was achieved in E. coli, a molecular mass of 35 kD GST fusion protein was purified, whose molecular mass and purity were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).


CONCLUSIONS
The expression vector for intracellular domain of hRAGE has been successfully constructed and efficient expression of the fusion protein is obtained, which can be of value for further studies.
Zhao
Shan-chao
SC
Key Laboratory of Shock and Microcirculation of PLA, First Military Medical University, Guangzhou 510515, China. yjiang@263.net
Liu
Jing-hua
JH
Li
Zhi-jie
ZJ
Qin
Qing-he
QH
Deng
Peng
P
Hou
Fan-fan
FF
Jiang
Yong
Y
D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase