This research sought to determine which induction condition resulted in the greatest GST-GFP fusion protein expression. It will hopefully serve as a guide for future researchers trying to produce their own recombinant protein containing GST and GFP-tags. The CDNB Enzyme Assay was used to determine the quantity of GST-GFP fusion protein present and tested three variables: IPTG concentration, duration, and temperature of induction. The findings showed that IPTG concentration, temperature, and induction duration all had a significant impact on protein expression. Induction temperatures of 20 °C and 25 °C showed better protein expression at IPTG concentrations of 1.0 mM IPTG over 0.1 mM IPTG. Induction durations of 20 hours and 5 hours were better than 3 hours. The 25 °C condition had the greatest protein expression, followed by 20 °C condition. In a follow-up experiment, using 1.0 mM IPTG and 20- hour induction duration, the 20 °C condition showed higher GST activity. Analysis of the 20 °C Western blots revealed the presence of possibly truncated/degraded fusion protein (not seen in the 25 °C Western blots). Future experimenters should use C-terminal GST-tags, as opposed to N-terminal GST-tags, to prevent accidental purification of truncated proteins in addition to the target protein. If this is not possible, then the recommendation is to use the 25 °C temperature for induction, as this temperature had similar GST-GFP fusion protein expression and resulted in much cleaner Western blots.
Bachelor of Science (B.S.)
College of Medicine
Burnett School of Biomedical Sciences
Vaccaro, Matthew J., "Expression Optimization of the GST-GFP Fusion Protein through the Alteration of Induction Conditions" (2023). Honors Undergraduate Theses. 1430.