Expression of a highly unstable and insoluble transcription factor in Escherichia coli: purification and characterization of the fork head homolog HNF3 alpha.
The HNF3 alpha transcription factor is highly enriched in adult hepatocytes and activates many liver-specific genes; its expression in hepatic primordia and neurogenic tissue implies a developmental role. HNF3 alpha is a member of a family of proteins, including fork head in Drosophila, which bind DNA at specific sites using a newly defined "winged helix" structural motif. We describe here the expression and purification of full-length HNF3 alpha containing an amino-terminal histidine tag and show that the protein binds DNA with dissociation constant in the sub-nanomolar range. The techniques used to isolate HNF3 alpha should be applicable to other members of the HNF3/fork head family which appear to be generally unstable and insoluble in Escherichia coli.
Zaret
K S
KS
Department of Molecular Biology, Cellular Biology and Biochemistry, Brown University, Providence, Rhode Island 02912, USA. zaret@brown.edu
Stevens
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics; D051858:Forkhead Transcription Factors; D015870:Gene Expression; D051541:Hepatocyte Nuclear Factor 3-alpha; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D014157:Transcription Factors / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
D004268:DNA-Binding Proteins; D051858:Forkhead Transcription Factors; C496501:Foxa1 protein, rat; D051541:Hepatocyte Nuclear Factor 3-alpha; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D004247:DNA