Expression, purification, and characterization of a carbohydrate-active enzyme: A research-inspired methods optimization experiment for the biochemistry laboratory.

Abstract

The development and implementation of research-inspired, discovery-based experiences into science laboratory curricula is a proven strategy for increasing student engagement and ownership of experiments. In the novel laboratory module described herein, students learn to express, purify, and characterize a carbohydrate-active enzyme using modern techniques and instrumentation commonly found in a research laboratory. Unlike in a traditional cookbook-style experiment, students generate their own hypotheses regarding expression conditions and quantify the amount of protein isolated using their selected variables. Over the course of three 3-hour laboratory periods, students learn to use sterile technique to express a protein using recombinant DNA in E. coli, purify the resulting enzyme via affinity chromatography and dialysis, analyze the success of their purification scheme via SDS-PAGE, assess the activity of the enzyme via an HPLC-based assay, and quantify the amount of protein isolated via a Bradford assay. Following the completion of this experiment, students were asked to evaluate their experience via an optional survey. All students strongly agreed that this laboratory module was more interesting to them than traditional experiments because of its lack of a pre-determined outcome and desired additional opportunities to participate in the experimental design process. This experiment serves as an example of how research-inspired, discovery-based experiences can benefit both the students and instructor; students learned important skills necessary for real-world biochemistry research and a more concrete understanding of the research process, while generating new knowledge to enhance the scholarly endeavors of the instructor.