Identification and characterization of endonuclein binding proteins: evidence of modulatory effects on signal transduction and chaperone activity
1 Department of Medical Biochemistry, Aarhus University, Ole Worms Allé 3, Building 1170, Aarhus, DK-8000 Aarhus C, Denmark
2 Department of Ophthalmology, Aalborg Hospital, Aarhus University Hospital, Hobrovej 10, DK-9000 Aalborg, Denmark
BMC Biochemistry 2009, 10:34 doi:10.1186/1471-2091-10-34Published: 22 December 2009
We have previously identified endonuclein as a cell cycle regulated WD-repeat protein that is up-regulated in adenocarcinoma of the pancreas. Now, we aim to investigate its biomedical functions.
Using the cDNA encoding human endonuclein, we have expressed and purified the recombinant protein from Escherichia coli using metal affinity chromatography. The recombinant protein was immobilized to a column and by affinity chromatography several interacting proteins were purified from several litres of placenta tissue extract. After chromatography the eluted proteins were further separated by two-dimensional gel electrophoresis and identified by tandem mass spectrometry. The interacting proteins were identified as; Tax interaction protein 1 (TIP-1), Aα fibrinogen transcription factor (P16/SSBP1), immunoglobulin heavy chain binding protein (BiP), human ER-associated DNAJ (HEDJ/DNAJB11), endonuclein interaction protein 8 (EIP-8), and pregnancy specific β-1 glycoproteins (PSGs). Surface plasmon resonance analysis and confocal fluorescence microscopy were used to further characterize the interactions.
Our results demonstrate that endonuclein interacts with several proteins indicating a broad function including signal transduction and chaperone activity.