Cloning and purification of functionally active Fas ligand interfering protein (FIP) expressed in Escherichia coli

• Authors: Pawel Wisniewski , Adam Master , Bozena Kaminska
• Languages of publication: EN

Abstracts

EN

This report presents purification and characterization of the extracellular domain of rat Fas protein, called FIP (FasL interfering protein), expressed as inclusion bodies in Escherichia coli. FIP was extracted from the inclusion bodies, solubilized with 8 M urea, purified by a single-step immobilized metal ion (Ni2+) affinity chromatography and refolded. SDS/PAGE and mass spectrometry analysis of the purified protein verified its purity. Fluorescence spectrum analysis showed that the refolding procedure caused structural changes which presumably might have led to oligomerization. The purified FIP has biological activities: it binds specifically soluble Fas ligand and protects human Jurkat lymphocytes against FasL-dependent apoptosis. This efficient procedure of FIP expression in E. coli and renaturation may be useful for production of therapeutically important proteins.

Keywords

EN

Fas ligand; interfering molecules; recombinant protein purification; Fas; apoptosis

• Publisher: Polish Biochemical Society
• Journal: Acta Biochimica Polonica
• Year: 2008
• Volume: 55
• Issue: 1
• Pages: 51-56

Dates

published

2008

accepted

2007-01-04

received

2007-10-05

revised

2007-12-04

(unknown)

2008-01-18

Contributors

author

Pawel Wisniewski

• Laboratory of Transcription Regulation, Department of Cell Biology, The Nencki Institute of Experimental Biology, Warszawa, Poland

author

Adam Master

• Laboratory of Transcription Regulation, Department of Cell Biology, The Nencki Institute of Experimental Biology, Warszawa, Poland

author

Bozena Kaminska

• Laboratory of Transcription Regulation, Department of Cell Biology, The Nencki Institute of Experimental Biology, Warszawa, Poland

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