Biological function of fusion protein ATF-PAI2CD

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

Research output: Contribution to journalArticle

4 Citations (Scopus)

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 - Aug 22 2003

Fingerprint

Plasminogen Activator Inhibitor 2
Fusion reactions
Polyamideimides
Urokinase-Type Plasminogen Activator
Proteins
Escherichia coli
Assays
Bearings (structural)
Chromogenics
Bacterial Proteins
Inclusion Bodies
Ion Exchange Chromatography
Chromatography
Radio
Washing
Sepharose
Polyacrylamide Gel Electrophoresis
Lung Neoplasms
Ion exchange
Milk

Keywords

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

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry

Cite this

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

Biological function of fusion protein ATF-PAI2CD. / Wang, Xia; Li, Ping; Zhang, Yu Qing; Hou, Min; Sun, Xinghui; 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 journalArticle

Wang, X, Li, P, Zhang, YQ, Hou, M, Sun, X, Tan, L & Zhu, YS 2003, 'Biological function of fusion protein ATF-PAI2CD', Acta Biochimica et Biophysica Sinica, vol. 35, no. 7, pp. 624-628.
Wang X, Li P, Zhang YQ, Hou M, Sun X, Tan L et al. Biological function of fusion protein ATF-PAI2CD. Acta Biochimica et Biophysica Sinica. 2003 Aug 22;35(7):624-628.
Wang, Xia ; Li, Ping ; Zhang, Yu Qing ; Hou, Min ; Sun, Xinghui ; Tan, Li ; Zhu, Yun Song. / Biological function of fusion protein ATF-PAI2CD. In: Acta Biochimica et Biophysica Sinica. 2003 ; Vol. 35, No. 7. pp. 624-628.
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