Use of Green Fluorescent Protein (GFP) to illustrate intra- and extracellular expression of recombinant proteins in the yeast Pichia pastoris

Abstract

At the Federal University of São Carlos (São Carlos, SP, Brazil), third-year biotechnology undergraduate students are required to take the theoretical and practical course "Expression and Purification of Recombinant Proteins”. The course includes laboratory exercises that show how to express proteins in the Pichia pastoris and Escherichia coli systems, as well as how to purify proteins using affinity chromatography. This hands-on course used the green fluorescente protein (GFP) as a model to demonstrate intracellular and extracellular protein expression in the yeast P. pastoris, aiming to illustrate the role of the signal peptide in protein targeting and the potential reasons for differences in GFP expression levels between different P. pastoris transformants. To achieve this, we cloned the GFP coding sequence into the pPICZA and pPICZaA vectors, enabling high-level methanol-induced recombinant expression of this protein. We divided the students into two groups in class, one with intracellular expression and the other with extracelular expression, and taught them the procedure for recombinant protein expression in P. pastoris. After conducting laboratory experiments, we discussed and compared the results of protein expression and signaling between the groups, highlighting some of the course's key topics. At the end, they were asked to respond to a questionnaire assessing the course's overall quality and the degree to which it contributed to their understanding of the topics on a 1-5 Likert scale. According to the answers, the content comprehension was generally good. The most effective assimilation topic was the one regarding the expression systems (4.8 for E. coli and 4.83 for P. pastoris) in comparison to the contextualization of the GFP scientific history (4.22). Our in-class discussion and questionnaire analysis led us to believe we successfully achieved our objectives. This result encourages the use of this methodology in undergraduate classes.