Production of an Improved Vector for Synthesis and Purification of Eukaryotic Transcription Factors (GAL4-VP16, GAL4-AD and GAL4-D)

The main goal in conducting this series of experiments is to accomplish the construction of an expression vector that contains the coding regions for the GAL4 series transcription factors. This expression vector should possess special characteristics including a histidine tag that helps to easily locate the vector. The other main characteristic is a T7 promoter that allows for a high induction level upon addition of IPTG. The original pET21d expression vector was used in these experiments because it possesses these two intrinsic features. A sub-cloning process of the coding regions of the GAL4 series transcription factors (GAL4-AD, GAL4-D and GAL4-VP16) into the original pET21d expression vector resulted in the pET2Id-GAL4 expression vector. The coding regions for the GAL4-AD and GAL4-D transcription factors were extracted from a pGEX-cs vector by Polymerase Chain Reaction (PCR) amplification with specially designed primers. The GAL4-VP16 transcription factor was extracted from the pJL2 plasmid. In the case of the GAL4-VP16 transcription factor, the sequence was not initially available and experimental sequencing of the transcription factor was necessary to acquire enough information about the sequence to design the primers for the PCR amplification