Expression, purification, and characterization of the 4 zinc finger region of human tumor suppressor WT1.
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 betabetaalpha 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-TGG-GCG-3' confirmed that 6HIS-ZN-wt1 has higher DNA binding affinity than 6HIS-ZN+wt1.
Nurmemmedov
Elmar
E
Department of Molecular Biophysics, Lund University, Chemical Center, Box 124, SE 221 00, Lund, Sweden. Elmar.Nurmemmedov@mbfys.lu.se
Thunnissen
Marjolein
M
D017398:Alternative Splicing / Q000235:genetics; D002853:Chromatography, Liquid / Q000379:methods; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D011485:Protein Binding / Q000235:genetics; D017510:Protein Folding; D020033:Protein Isoforms / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D025721:WT1 Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D016335:Zinc Fingers / Q000235:genetics
D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D025721:WT1 Proteins