Expression of a zinc-binding domain of boar spermatidal transition protein 2 in Escherichia coli.
Transition protein 2 (TP2; 137 amino acid residues) from boar late spermatid nuclei has three potential zinc finger motifs in the N-terminal 34 region. Gel shift assays revealed that boar TP2 recognized a CpG island sequence in a zinc-dependent manner. However, there was some nonspecific recognition of the oligonucleotide. Then, we constructed the expression system of zinc-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. TP2Z was expressed upon induction with 1 mM isopropylthiogalactoside and extracted with acid including 0.71 M 2-mercaptoethanol. TP2Z was purified by ion-exchange chromatography on Fractogel EMD SO(-)(3) and HPLC on Nucleosil 300 7C18 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.
Sato
H
H
Graduate School of Science and Technology, Chiba University, Chiba, Chiba, 263-8522, Japan.
Akama
K
K
Kojima
S
S
Miura
K
K
Sekine
A
A
Nakano
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D018899:CpG Islands; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D008297:Male; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013087:Spermatids / Q000737:chemistry* / Q000378:metabolism*; D013552:Swine; D013997:Time Factors; D016335:Zinc Fingers / Q000235:genetics*
D009687:Nuclear Proteins; D011994:Recombinant Proteins