Construction, synthesis, and expression of a gene encoding a protein with two DNA-binding motifs

A gene encoding a protein with two DNA-binding motifs was constructed, synt hesized, and expressed in Escherichia coli. The gene nucleotide sequence co ntains codons predominant in genes efficiently expressed in E. coli. It als o has restriction sites at the beginning and at the end of the DNA-binding motifs. This facilitates replacing the DNA-binding motifs as well as alteri ng the connecting sequence. Six successive histidine codons were introduced into the gene region encoding the C-terminal part of the protein to expedi te protein isolation. The DNA fragments corresponding to the N- and C-termi nal protein domains were synthesized and cloned in pGEM7z(f+) between the X baI/XhoI and XhoI/BamHI sites, respectively. The full-sized gene sequence w as isolated and cloned in the modified expression vector pTZ19R. The chimer ic gene including the translation initiation region, four codons for the N- terminal amino acids of the E. coil protein OmpA, and the synthetic DNA fra gment was expressed under the he promoter. The expression product of 110 aa was isolated using Quiagen adsorbents.