Abstract

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.

Thesis Completion

2023

Semester

Spring

Thesis Chair/Advisor

Borgon, Robert

Degree

Bachelor of Science (B.S.)

College

College of Medicine

Department

Burnett School of Biomedical Sciences

Degree Program

Biomedical Sciences

Language

English

Access Status

Open Access

Release Date

5-15-2023

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