Expression, purification and characterization of the homeodomain of rat ISL-1 protein.
Isl-1 is a member of a family of Homeodomains containing proteins that possess an N-terminal pair of zinc binding LIM domains. The Isl-1 gene in rat codes for a protein that binds to the insulin gene enhancer and is also involved in regulation of amylin and proglucagon genes. A DNA sequence coding for 66 amino acid residues containing the C-terminal homeodomain fragment of Isl-1 was expressed as a soluble protein in Escherichia coli. Here, we describe a procedure which allows the rapid native purification of recombinant homeodomain protein fused to an N-terminal tag of six histidines. The purified homeodomain showed DNA-binding activity to its cognate DNA sequence. An enhanced binding activity is observed in the presence of a reducing agent in electrophoretic mobility shift assays. The DNA binding was further characterized by circular dichroism spectroscopy. Addition of DNA to the homeodomain did not change the overall secondary structure content, but the thermal and chemical denaturing profiles were altered. A stabilization of the secondary structure was observed upon DNA binding. The free energy of unfolding at 23 degrees C was 7 kJ mol(-1) in absence of DNA and 29 kJ mol(-1) in the presence of DNA.
Behravan
G
G
Department of Medical Biochemistry and Biophysics, Umeå University, Sweden.
Lycksell
P O
PO
Larsson
G
G
D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004742:Enhancer Elements, Genetic; D015870:Gene Expression; D006639:Histidine / Q000235:genetics; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D007328:Insulin / Q000235:genetics; D060850:LIM-Homeodomain Proteins; D009419:Nerve Tissue Proteins; D017510:Protein Folding; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification; D013816:Thermodynamics; D014157:Transcription Factors; D015032:Zinc / Q000378:metabolism
D018398:Homeodomain Proteins; D007328:Insulin; D060850:LIM-Homeodomain Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; C063843:insulin gene enhancer binding protein Isl-1; D006639:Histidine; D004247:DNA; D015032:Zinc