Biological function of fusion protein ATF-PAI2CD

Xia Wang, Ping Li, Yu Qing Zhang, Min Hou, Xing Hui Sun, Li Tan, Yun Song Zhu

Research output: Contribution to journalArticle

4 Scopus citations

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 languageEnglish (US)
Pages (from-to)624-628
Number of pages5
JournalActa Biochimica et Biophysica Sinica
Volume35
Issue number7
StatePublished - 2003

Keywords

  • Expression
  • Fusion protein
  • Purification
  • Radio ligand receptor binding assay

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry

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    Wang, X., Li, P., Zhang, Y. Q., Hou, M., Sun, X. H., Tan, L., & Zhu, Y. S. (2003). Biological function of fusion protein ATF-PAI2CD. Acta Biochimica et Biophysica Sinica, 35(7), 624-628.