Implementing a Hybrid Expression Method That Allows Upper-Division Biochemistry Lab Students To Engage in a Full Protein Production Experience While Allowing Ample Time for Characterization Experiments

Abstract

Protein structure, function, and signaling are a large portion of biochemistry. Because of this, proteins are often used as model systems in biochemistry laboratory courses, where a course-long project might comprise protein expression, purification, and characterization. Two common protein expression methods are isopropyl β-d-1-thiogalactopyranoside (IPTG) induction, which utilizes easy-to-make media but requires extensive cell-growth monitoring that is time-intensive, and autoinduction, which employs multicomponent media that are time-consuming to make but require no cell-growth monitoring. A protein expression method that is a hybrid of IPTG induction and autoinduction is presented. The hybrid method utilizes the medium of IPTG induction and the no-cell-growth-monitoring induction process of autoinduction, saving hands-on time in the protein expression phase to allow more time for protein characterization while still having students execute each step.

Keywords: Biochemistry; Enzymes; Graduate Education/Research; Hands-On Learning/Manipulatives; Laboratory Instruction; Proteins/Peptides; Upper-Division Undergraduate.

Figure 1.

Schematic comparison of three expression methods: IPTG induction, autoinduction, and TB autoinduction.

Figure 2.

Coomassie-stained 14% SDS-PAGE of CaM elutions produced by TB autoinduction. kDa indicates the molecular weight in kilodaltons of each protein standard in the first lane. M: marker (proteins of known molecular weight). E1–E7: elutions 1–7. Elution buffer (50 mL) was applied to the column, and each collected elution was 5 mL. CaM fully elutes between 5 and 25 mL of elution buffer (E2–E5). Samples of each elution were diluted 1:10, and 5 μL of each diluted elution was loaded in the gel. CaM is 16.8 kDa and is the visible band in each of the lanes marked E2–E5, as determined by mass spectrometry.

Figure 3.

Percentage of protein produced by each expression method relative to IPTG induction for the same protein. CaM, PEP19, and CaN were each expressed by IPTG induction, autoinduction, and TB autoinduction. The amount of protein produced for each protein and method is shown here as a percentage of IPTG induction for the same protein.