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http://dx.doi.org/10.4014/jmb.0905.05021

Purification of Caudal-Related Homeodomain Transcription Factor and Its Binding Characterization  

Jeong, Mi-Suk (Department of Molecular Biology, College of Natural Sciences, Pusan National University)
Hwang, Eun-Young (Department of Molecular Biology, College of Natural Sciences, Pusan National University)
Kim, Hyun-Tae (Department of Molecular Biology, College of Natural Sciences, Pusan National University)
Yoo, Mi-Ae (Department of Molecular Biology, College of Natural Sciences, Pusan National University)
Jang, Se-Bok (Department of Molecular Biology, College of Natural Sciences, Pusan National University)
Publication Information
Journal of Microbiology and Biotechnology / v.19, no.12, 2009 , pp. 1557-1564 More about this Journal
Abstract
Human CDX2 is known as a caudal-related homeodomain transcription factor that is expressed in the intestinal epithelium and is important in differentiation and maintenance of the intestinal epithelial cells. The caudal-related homeobox proteins bind DNA according to a helix-turn-helix structure, thereby increasing the structural stability of DNA. A cancer-tumor suppressor role for Cdx2 has been shown by a decrease in the level of the expression of Cdx2 in colorectal cancer, but the mechanism of transcriptional regulation has not been examined at the molecular level. We developed a large-scale system for expression of the recombinant, novel CDX2, in Escherichia coli. A highly purified and soluble CDX2 protein was obtained in E. coli strain BL21(DE3)RIL and a hexahistidine fusion system using Ni-NTA affinity column, anion exchange, and gel filtration chromatographies. The identity and secondary structure of the purified CDX2 protein were confirmed by MALDI-TOF MS, Western blot, and a circular dichroism analyses. In addition, we studied the DNA-binding activity of recombinant CDX2 by ELISA experiment and isolated human CDX2-binding proteins derived from rat cells by an immobilized GST-fusion method. Three CDX2-binding proteins were found in the gastric tissue, and those proteins were identified to the homeobox protein Hox-D8, LIM homeobox protein 6, and SMC1L1 protein.
Keywords
Caudal-related homeodomain protein; cloning; overexpression; purification; binding; human CDX2;
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