Academic Awards 2025 booklet

21 The Expression and Characterization of Protein M Mutants for Improved Dissociation Kinetics Continuous biosensors, such as glucose monitors used in diabetes care, have transformed real-time health monitoring by enabling immediate responses to changing biomarker levels. This approach is particularly valuable because it allows treatment to be administered as soon as it is needed, potentially preventing severe complications. However, many current biosensing technologies are not compatible with small molecules and rely on complex, multi-step procedures that limit their use in a continuous manner. To address this, a new biosensor concept was proposed that uses a single antibody and an antibody-binding protein to generate a measurable signal. Protein M is a promising candidate due to its universal antibody- binding ability, but its binding to antibodies is too stable for dynamic, continuous sensing. This project aimed to engineer variants of Protein M with improved dissociation kinetics, capable of binding and releasing antibodies more easily. Six mutants were designed through site-directed mutagenesis, then expressed, purified, and analysed. None of the mutations affected the thermal stability of protein M. This work provides a strong foundation for future characterization using techniques such as surface plasmon resonance. Ultimately, it contributes to the development of continuous biosensors capable of real-time small molecule monitoring and timely, responsive medical intervention. 0 10 20 30 40 50 TK01 TK02 TK11 TK01-TR TK02-TR TK03-TR TK04-TR TK05-TR TK11-TR WT P42 41.1 43.1 42.6 42.5 43.5 45.1 44.0 44.7 42.4 43.4 43.6 Thermal Shift Assay Result Melting Temperature [°C] Protein Figure 1: A novel biosensing construct for continuous sensing; visible light emission depends on presence of a biomarker of interest. Protein M could be used as the competitor molecule (visualized in red) but its binding to antibodies is too strong for use in a dynamic system. Figure 2: All the mutant proteins have melting temperatures above 40 °C and are thus stable at room temperature. The original protein (WT) and a known mutant (P42) were included as controls.