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鉴定和表征核酸内切酶结合蛋白：对信号转导和伴侣活性的调节作用的证据。

Identification and characterization of endonuclein binding proteins: evidence of modulatory effects on signal transduction and chaperone activity.

机构信息

Department of Medical Biochemistry, Aarhus University, Ole Worms Allé 3, Building 1170, Aarhus, DK-8000 Aarhus C, Denmark.

出版信息

BMC Biochem. 2009 Dec 22;10:34. doi: 10.1186/1471-2091-10-34.

DOI:10.1186/1471-2091-10-34
PMID:20028516
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC2810291/
Abstract

BACKGROUND

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.

RESULTS

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), Aalpha 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 beta-1 glycoproteins (PSGs). Surface plasmon resonance analysis and confocal fluorescence microscopy were used to further characterize the interactions.

CONCLUSIONS

Our results demonstrate that endonuclein interacts with several proteins indicating a broad function including signal transduction and chaperone activity.

摘要

背景

我们之前已经鉴定出内核酸酶是一种细胞周期调控的 WD 重复蛋白，在胰腺腺癌中上调。现在，我们旨在研究其生物医学功能。

结果

使用编码人内核酸酶的 cDNA，我们使用金属亲和层析从大肠杆菌中表达和纯化了重组蛋白。将重组蛋白固定在柱上，通过亲和层析从几升胎盘组织提取物中纯化出几种相互作用的蛋白质。层析后，洗脱的蛋白质通过二维凝胶电泳进一步分离，并通过串联质谱鉴定。鉴定出的相互作用蛋白包括：Tax 相互作用蛋白 1（TIP-1）、Aalpha 纤维蛋白原转录因子（P16/SSBP1）、免疫球蛋白重链结合蛋白（BiP）、人 ER 相关 DNAJ（HEDJ/DNAJB11）、内核酸酶相互作用蛋白 8（EIP-8）和妊娠特异性β-1 糖蛋白（PSGs）。表面等离子体共振分析和共聚焦荧光显微镜用于进一步表征相互作用。

结论

我们的结果表明，内核酸酶与几种蛋白质相互作用，表明其具有广泛的功能，包括信号转导和伴侣活性。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/45e0/2810291/1aa52f125cec/1471-2091-10-34-6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/45e0/2810291/32da9badbdad/1471-2091-10-34-1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/45e0/2810291/fbc2148edb53/1471-2091-10-34-2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/45e0/2810291/a1d0188c75ea/1471-2091-10-34-3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/45e0/2810291/e5a0314e7a76/1471-2091-10-34-4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/45e0/2810291/486857913f9a/1471-2091-10-34-5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/45e0/2810291/1aa52f125cec/1471-2091-10-34-6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/45e0/2810291/32da9badbdad/1471-2091-10-34-1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/45e0/2810291/fbc2148edb53/1471-2091-10-34-2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/45e0/2810291/a1d0188c75ea/1471-2091-10-34-3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/45e0/2810291/e5a0314e7a76/1471-2091-10-34-4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/45e0/2810291/486857913f9a/1471-2091-10-34-5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/45e0/2810291/1aa52f125cec/1471-2091-10-34-6.jpg

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