Optimizing GFP-GST Rearranged Fusion Protein Expression at 20℃ in E. coli BL21 cells

Authors

Alyssa R. Ramirez, University of Central FloridaFollow

Streaming Media

Files

Course Code

PCB

Course Number

4943

Faculty/Instructor

Nicole Verity, Dr. Robert Borgon

Faculty/Instructor Email

nicole.verity@ucf.edu, robert.borgon@ucf.edu

About the Author

Hello! My name is Alyssa Ramirez, a senior at the University of Central Florida with a major in Biomedical Sciences and minor in Medical Anthropology. I am grateful to Professor Nicole Verity and Dr. Robert Borgon for supporting me throughout this project through their guidance and mentorship. I would also like to acknowledge the contributions of Mohammad Jamous, a Biomedical Sciences student who assisted in designing the project and collecting data through the PILOT course as well.

Abstract, Summary, or Creative Statement

Link to video: https://ucf.zoom.us/rec/share/aBJX0KJknmMOUuFrSjovlwOuvT4kLbg-iwCStJrh-oajoZtUlOmJwOVl3hgj0Pt5.oHtFyE-wSzomA05f?startTime=1712587387000

Recording password: Z.cZjgz4

This project seeks to expand upon the findings in Vaccaro’s (2023) study of induction conditions for maximal glutathione-S-transferase green fluorescent protein (GST-GFP) in Escherichia coli. After investigating induction parameters such duration, temperature, and inducer concentration, Vaccaro found that optimal protein expression occurred after inducing growth for 20 hours at 25℃ with an inducer concentration of 1.0 mM. Nevertheless, significant background protein expression was identified at an induction temperature of 20℃ which was attributed to degraded GST-GFP expression. Our research intends to optimize this protocol by utilizing a GFP-GST rearranged fusion protein vector to avoid this truncated protein expression. We aim to accomplish this by expressing the rearranged vector in E. coli, obtaining the GFP-GST protein, measuring reaction velocity, and purifying via affinity and ion exchange chromatography. We will then visualize the protein via SDS-PAGE and Western blotting procedures. We anticipate that rearranging GST-GFP and inducing expression at 20℃ for 20 hours with an inducer concentration of 1.0 mM will reduce the Western blot background expression seen in Vaccaro’s (2023) original work, thus improving the GST-GFP expression and purification protocols for use by researchers attempting to optimize their own protocols involving this recombinant fusion protein.

Additional Resources

References include:

1. Vaccaro, Matthew J., "Expression Optimization of the GST-GFP Fusion Protein through the Alteration of Induction Conditions" (2023). Honors Undergraduate Theses. 1430. https://stars.library.ucf.edu/honorstheses/1430.

1. Borgon, R and Verity, N. (2019). Quantitative Biological Methods. Hayden-McNeil.

Recording password: Z.cZjgz4

Keywords

protein, expression, purification, recombinant, E. coli, green fluorescent protein, glutathione-S-transferase, molecular biology, microbiology

Recommended Citation

Ramirez, Alyssa R., "Optimizing GFP-GST Rearranged Fusion Protein Expression at 20℃ in E. coli BL21 cells" (2023). High Impact Practices Student Showcase Spring 2024. 16.
https://stars.library.ucf.edu/hip-2024spring/16