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 gen e in rat codes for a protein that binds to the insulin gene enhancer a nd is also involved in regulation of amylin and proglucagon genes, ii DNA sequence coding for 66 amino acid residues containing the C-termin al homeodomain fragment of Isl-l was expressed as a soluble protein in Escherichia coli, Here, we describe a procedure which allows the rapi d native purification of recombinant homeodomain protein fused to an N -terminal tag of six histidines, The purified homeodomain showed DNA-b inding activity to its cognate DNA sequence, An enhanced binding activ ity is observed in the presence of a reducing agent in electrophoretic mobility shift assays, The DNA binding was further characterized by c ircular dichroism spectroscopy, Addition of DNA to the homeodomain did not change the overall secondary structure content, but the thermal a nd chemical denaturing profiles were altered, A stabilization of the s econdary structure was observed upon DNA binding. The free energy of u nfolding at 23 degrees C was 7 kJ mol(-1) in absence of DNA and 29 kJ mol(-1) in the presence of DNA.