Abstract
To express the fusion protein ATF-PAI2CD (urokinase-type plasminogen activator amino terminal fragment-plasminogen activator inhibitor type 2 with the region inter C and D helices deleted) gene in E. coli and determine the biological characterization of fusion protein ATF-PAI2CD, the cDNA fragment encoding ATF-PAI2CD was cloned into the expression vector pLY-4 and transformed into E. coli JF1125. After temperature induction, the expression amount of ATF-PAI2CD account for 15% of total bacterial protein. The result was confirmed by Western blot. ATF-PAI2CD protein was isolated and purified by washing and solubilization of inclusion body, renaturation and ion exchange chromatography. The final product displayed a single band with a corresponding molecular weight 62 kD in SDS-PAGE. The purity was over 90%, the protein yield was 50% and the specific activity was 12 000 IU/mg. The PAI activity was measured by chromogenic assay. The purified fusion protein inhibited urokinase-type plasminogen activator as measured by milk-agarose plate assay, and bound to human lung cancer cells via uPA receptor (uPAR), which was confirmed by radio competition experiments. The results indicate that the biological characteristics of ATF-PAI2CD were very similar to those of the wide type PAI-2 (or mutants PAI-2, PAI-2CD) and to pro-uPA in binding to uPAR-bearing cells.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 624-628 |
| Number of pages | 5 |
| Journal | Acta Biochimica et Biophysica Sinica |
| Volume | 35 |
| Issue number | 7 |
| State | Published - Aug 22 2003 |
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Keywords
- Expression
- Fusion protein
- Purification
- Radio ligand receptor binding assay
ASJC Scopus subject areas
- Biophysics
- Biochemistry
Cite this
Biological function of fusion protein ATF-PAI2CD. / Wang, Xia; Li, Ping; Zhang, Yu Qing; Hou, Min; Sun, Xing Hui; Tan, Li; Zhu, Yun Song.
In: Acta Biochimica et Biophysica Sinica, Vol. 35, No. 7, 22.08.2003, p. 624-628.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Biological function of fusion protein ATF-PAI2CD
AU - Wang, Xia
AU - Li, Ping
AU - Zhang, Yu Qing
AU - Hou, Min
AU - Sun, Xing Hui
AU - Tan, Li
AU - Zhu, Yun Song
PY - 2003/8/22
Y1 - 2003/8/22
N2 - To express the fusion protein ATF-PAI2CD (urokinase-type plasminogen activator amino terminal fragment-plasminogen activator inhibitor type 2 with the region inter C and D helices deleted) gene in E. coli and determine the biological characterization of fusion protein ATF-PAI2CD, the cDNA fragment encoding ATF-PAI2CD was cloned into the expression vector pLY-4 and transformed into E. coli JF1125. After temperature induction, the expression amount of ATF-PAI2CD account for 15% of total bacterial protein. The result was confirmed by Western blot. ATF-PAI2CD protein was isolated and purified by washing and solubilization of inclusion body, renaturation and ion exchange chromatography. The final product displayed a single band with a corresponding molecular weight 62 kD in SDS-PAGE. The purity was over 90%, the protein yield was 50% and the specific activity was 12 000 IU/mg. The PAI activity was measured by chromogenic assay. The purified fusion protein inhibited urokinase-type plasminogen activator as measured by milk-agarose plate assay, and bound to human lung cancer cells via uPA receptor (uPAR), which was confirmed by radio competition experiments. The results indicate that the biological characteristics of ATF-PAI2CD were very similar to those of the wide type PAI-2 (or mutants PAI-2, PAI-2CD) and to pro-uPA in binding to uPAR-bearing cells.
AB - To express the fusion protein ATF-PAI2CD (urokinase-type plasminogen activator amino terminal fragment-plasminogen activator inhibitor type 2 with the region inter C and D helices deleted) gene in E. coli and determine the biological characterization of fusion protein ATF-PAI2CD, the cDNA fragment encoding ATF-PAI2CD was cloned into the expression vector pLY-4 and transformed into E. coli JF1125. After temperature induction, the expression amount of ATF-PAI2CD account for 15% of total bacterial protein. The result was confirmed by Western blot. ATF-PAI2CD protein was isolated and purified by washing and solubilization of inclusion body, renaturation and ion exchange chromatography. The final product displayed a single band with a corresponding molecular weight 62 kD in SDS-PAGE. The purity was over 90%, the protein yield was 50% and the specific activity was 12 000 IU/mg. The PAI activity was measured by chromogenic assay. The purified fusion protein inhibited urokinase-type plasminogen activator as measured by milk-agarose plate assay, and bound to human lung cancer cells via uPA receptor (uPAR), which was confirmed by radio competition experiments. The results indicate that the biological characteristics of ATF-PAI2CD were very similar to those of the wide type PAI-2 (or mutants PAI-2, PAI-2CD) and to pro-uPA in binding to uPAR-bearing cells.
KW - Expression
KW - Fusion protein
KW - Purification
KW - Radio ligand receptor binding assay
UR - http://www.scopus.com/inward/record.url?scp=0042071206&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0042071206&partnerID=8YFLogxK
M3 - Article
C2 - 12883632
AN - SCOPUS:0042071206
VL - 35
SP - 624
EP - 628
JO - Acta Biochimica et Biophysica Sinica
JF - Acta Biochimica et Biophysica Sinica
SN - 1672-9145
IS - 7
ER -