Expression and characterization of recombinant mouse and human DUSP2 protein

Abstract

Background: This master's thesis focuses on the DUSP2 phosphatase protein. This protein plays a vital role in regulating cellular signaling pathways, including MAPK signaling involving JNK1, p38α, and ERK2. By better understanding DUSP2's physiological role within these pathways, we can gain a deeper understanding of cellular mechanisms, disease mechanisms, and perhaps even pave the way for new drugs to be developed. Method: The study aimed to optimize expression conditions and characterize both mouse and human DUSP2 proteins. The production of the desired protein involves three essential steps: molecular cloning, expression, and purification. The full coding sequences of mouse and human DUSP2 were cloned into an appropriate expression vector using a bacterial expression system. The resulting vector was transformed into a suitable host cell to produce the desired protein. Subsequent steps included purifying cell lysates through affinity chromatography, with the presence of recombinant GST-DUSP2 protein bands confirmed via SDS-PAGE analysis. Results: The findings of this study indicate the successful expression and purification of both mouse and human GST-DUSP2 proteins. Characterization of the protein included SDS-PAGE analysis, quantification, and enzymatic tests, such as phosphatase assays, to assess protein activity. However, despite multiple attempts, active human DUSP2 protein was not obtained, while the mouse DUSP2 displayed activity. Conclusion: In summary, this master's thesis provides insights into the challenges encountered in expressing and characterizing recombinant human GST-DUSP2 protein. While the study did not achieve the desired outcome of obtaining active human DUSP2 protein, it contributes to our understanding of the difficulties associated with human DUSP2 expression.