Purification of caudal-related homeodomain transcription factor and its binding characterization.
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 the 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 chromatography. 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.
Jeong
Mi Suk
MS
Department of Molecular Biology, College of Natural Sciences, Pusan National University, Busa, Korea.
Hwang
Eun Young
EY
Kim
Hyun Tae
HT
Yoo
Mi-Ae
MA
Jang
Se Bok
SB
D000595:Amino Acid Sequence; D000818:Animals; D000071616:CDX2 Transcription Factor; D018929:Cell Culture Techniques; D018797:Cell Cycle Proteins / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005770:Gastrointestinal Neoplasms / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D018398:Homeodomain Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D060850:LIM-Homeodomain Proteins; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D000098550:Structural Maintenance of Chromosome Protein 1
D000071616:CDX2 Transcription Factor; C491270:CDX2 protein, human; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; C549598:HOXD8 protein, rat; D018398:Homeodomain Proteins; C112960:LIM homeobox protein 8; D060850:LIM-Homeodomain Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D000098550:Structural Maintenance of Chromosome Protein 1; D004247:DNA