Expression of a zinc-binding domain of boar spermatidal transition protein2 in Escherichia coli

Transition protein 2 (TP2; 137 amino acid residues) from boar late spermati d nuclei has three potential zinc finger motifs in the N-terminal 3/4 regio n. Gel shift assays revealed that boar TP2 recognized a CpG island sequence in a zinc-dependent manner. However, there was some nonspecific recognitio n of the oligonucleotide. Then, we constructed the expression system of zin c-binding domain of TP2 (TP2Z) (residues 1-103) in Escherichia coli. Double -stranded DNA fragments encoding TP2Z were synthesized as 18 fragments with 103 residues, annealed, and cloned into the expression plasmid pET11d. TP2 Z was expressed upon induction with 1 mM isopropylthiogalactoside and extra cted with acid including 0.71 M 2-mercaptoethanol. TP2Z was purified by ion -exchange chromatography on Fractogel EMD SO3- and HPLC on Nucleosil 300 7C 18 and on Diol-120. Atomic absorption and CD spectroscopy showed that TP2Z bound three atoms of zinc per molecule of the protein and underwent a zinc- dependent conformational change in a manner similar to that for intact TP2. Gel shift assays indicated that TP2Z recognized a CpG island sequence more specifically than intact TP2 and that the specificity is dependent on zinc . (C) 1999 Academic Press.