Expression, Purification
and Functional Identification of Extracellular Part of Discoidin Domain
Receptor 2
WANG Ji-Cun, LIU Xin-Ping, NIE Xiao-Yan, WU Guo-Qiang, ZHANG
Wen-Hong, SHEN Lan, YAO Li-Bo*
( Department of Biochemistry and Molecular Biology, The fourth Military
Medical University, Xi’an 710032, China )
Abstract Discoidin
domain receptor 2 (DDR2) is a new type of receptor tyrosine kinases, and was
thought to be involved in the metastasis of some tumors. Its ligand is
fibrillar collagen. The activation of DDR2 induced by collagen mediates the
over-expression of matrix metalloproteinase 1 (MMP-1) in cells. A
specificinhibitor of DDR2 was necessary for the study of DDR2 function.
Theoretically, a soluble receptor could possibly be used as specific inhibitor
for the native receptor on cell membrane. In this report, a fragment (DB) of
extracellular part of DDR2 was cloned and expressed for the use as potential
inhibitor. This DB fragment corresponded to the polypeptide from the 23rd amino
acid residue to the 293rd amino acid residue of DDR2. The fragment was
amplified by RT-PCR from human lung cancer tissue, and the product was cloned
into pMD18-T vector. After identification by sequence analysis, the fragment
was sub-cloned into pGEX-4T-1 vector. Fusion protein of GST-DB was expressed in
JM109 E.coli cells as expected and the soluble part accounted for
about 13% of the total fusion protein. The soluble fusion protein was then
purified with glutathione affinity resin, and GST-DB with purity of 86.1% was
obtained. Competitive combination inhibitory test showed that the purified
GST-DB inhibited the interaction between collagen II and DDR2 on the surface of
RA synovial fibroblasts. Zymography analysis showed that the level of MMP-1 of
both NIH 3T3 cell and RA synovial fibroblasts with collagen II-stimulation
decreased after adding GST-DB fusion protein. The results indicated that the
fusion protein GST-DB could inhibit the function of DDR2 on cells, and DDR2
might mediate collagen II-induced over-expression of MMP-1 in these cells.
Key words discoidin domain receptor 2； extracellular part； expression； inhibitor； matrix metalloproteinase 1
*Corresponding author： Tel, 86-29-3374513； e-mail, [email protected]