Researcher(s)
- Timothy Yarnall, Biochemistry, University of Delaware
Faculty Mentor(s)
- Shara Compton, Biochemistry, University of Delaware
Abstract
Protein purification techniques and enzyme kinetics are essential to a modern laboratory curriculum for students pursuing careers in biochemistry. A novel experimental module for the upper-division undergraduate biochemistry laboratory has been adapted from research projects originating from the Schmitz Lab at University of Delaware. This developing multi-week student experiment investigates the degradation of green fluorescent protein (GFP) by ClpXP protease. ClpXP, composed of ATP-dependent ClpX unfoldase associated with proteolytic ClpP, is a molecular machine responsible for the disassembly of unwanted proteins in cells. The brightly-colored GFP substrate was expressed in E. coli and lysed by ultrasonication to produce a crude sample for students to purify by Ni2+-NTA affinity chromatography. ClpP and ClpX enzymes were overexpressed in E. coli and purified by both affinity and ion exchange chromatography. Following these experiments, the purified enzyme samples were prepared for student use in kinetic analysis. A fluorescence-based assay was adapted for students to determine Michaelis-Menten kinetic parameters for proteolysis of GFP. The GFP-ClpXP purification and kinetics module will be introduced to undergraduate biochemistry students enrolled in the lab course starting in Fall 2024. This lab module will also be supplemented with bioinformatics-based pre-lab exercises and interactive lecture activities allowing students to deepen their understanding of protein structure and function from the armchair. This is all in tandem with laboratory activities that will illustrate protein structure-function relationships explored in lecture courses, connect to current research in the Department of Chemistry and Biochemistry, and provide students with hands-on experience with fundamental modern biochemical techniques.