Cloning and Expression of the cDNA Encoding Human Tissue Inhibitor of Metalloproteinase-3 and Its Inhibition on Angiogenesis.
We have isolated the cDNA encoding the mature tissue inhibitor of metalloproteinase-3 (TIMP-3) by using RT-PCR method with total RNA extracted from human fresh placenta. The result of sequencing indicated that the mature TIMP-3 polypeptide contained 188 aa residues, including 12 conserved cysteinyl residues in the TIMP family. The expression plasmid pET-TIMP3 was constructed by inserting TIMP-3 cDNA into plasmid pET-24 (a(+)) containing T7 promoter and transformed into E. coli BL21 (DE3). An expression strain BLTIMP3 was selected. SDS-PAGE analysis revealed that the human TIMP-3 protein was highly expressed and accumulated up to above 30% of the total bacterial proteins in the form of inclusion body after induced by 1 mmol/L IPTG for 3--5 h. The partially purified and renatured TIMP-3 can significantly inhibit angiogenesis as shown by CAM assay.
Sun
Jian-Xin
JX
Shanghai Institute of Biochemistry, the Chinese Academy of Sciences, Shanghai 200031, China.
Zhang
Chuan-Xi
CX
Chen
Zhen-Zhen
ZZ
Wu
Xiang-Fu
XF