Wilm's Tumor gene 1 (WT1) encodes a zinc finger protein with four distinct splice isoforms. WT1 has a critical role in genesis of various cancer types both at the DNA/RNA and the protein level. The zinc-finger DNA-binding capacity of the protein is located in the C-terminal domain. Two recombinant proteins, 6HIS-ZN-(wt1) and 6HIS-ZN+(wt1), corresponding to two alternative splice variants of the C-terminal regions of human WT1 (-KTS) and WT1 (+KTS), respectively, were over-expressed with hexa-histidine fusion tags in inclusion bodies in Escherichia coli for crystallization studies. A combination of Ni2+-NTA affinity and size-exclusion chromatography was applied for purification of the proteins in denaturing conditions. The effects of various buffers, salts and other additives were scrutinized in a systematic screening to establish the optimal conditions for solubility and refolding of the recombinant WT1 proteins. Circular dichroism analysis revealed the expected beta beta alpha content for the refolded proteins, with a notable degradation of the alpha-helical segment in the DNA-free state. Electrophoretic mobility shift assay with double-stranded DNA containing the double Egr1 consensus site 5'-GCG-T GG-GCG-3' confirmed that 6HIS-ZN-(wt1) has higher DNA binding affinity than 6HIS-ZN+(wt1). (c) 2005 Elsevier Inc. All rights reserved.