PCR constructs of approximately 768-bp in length were designed for integration into the pET-32a(+) plasmid which carries His-tag, S-tag and trx-tag sequences. Protein expression of this transcription factor was induced in Escherichia coli BL21-DE3-transformed cells through heat shock using three different temperatures.
Protein isolation and SDS-PAGE analysis showed that 28oC was the optimal temperature for induction of protein expression, generating approximately 640 ug per extraction. Western blot analysis determined that the AP/ERF transcription factor was isolated, with an approximate size of 50 kDa.
Electron mobility shift assay (EMSA) results showed that the cis-element core CCAAC was necessary for the binding of the DRE element, with the substitution of the second or fifth position (C nucleotide) playing a critical role in its binding capacity.