The transcription factor Sp1 is a regulator of TATA-less genes. It belongs to the Cys(2)-His(2) zinc finger domain-containing family. A zebrafish cDNA encoding a peptide homologous to mammalian Sp1 was cloned and inserted into a pET43.1a vector and expressed in Escherichia coil Rosetta (DE3) cells as a Nus-His-tag fusion protein. After induction with isopropyl thiogalactoside, the protein was purified with a Ni-Sepharose column, and approximately 5-8 mg of pure protein was obtained per liter of culture. The primary sequence and the predicted partial tertiary structure of the potential recombinant zebrafish Sp1 protein are similar to those of human Sp1. The DNA affinity precipitation assay and dual-luciferase promoter activity assay further confirm the nature of the recombinant zebrafish Sp1 protein as a transcription factor. Our results show that zebrafish Sp1-like protein is structurally and functionally comparable to human Sp1. (C) 2010 Elsevier Inc. All rights reserved.