Poster Presentation The 47th Lorne Conference on Protein Structure and Function 2022

Engineering Fast-Responding Protein Biosensors (#316)

Cagla Ergun Ayva 1 , Zhong Guo 1 , Wayne Johnston 1 , Patricia Walden 1 , Kirill Alexandrov 1 2
  1. School of Biology and Environmental Science, Centre for Agriculture and the Bioeconomy, Queensland University of Technology, Brisbane, QLD, Australia
  2. CSIRO-QUT Synthetic Biology Alliance, ARC Centre of Excellence in Synthetic Biology, Institute of Health and Biomedical Innovation, Institute for Future Environments, Queensland University of Technology, Brisbane, QLD, Australia

Protein-based biosensors are essential tools for studying cellular mechanisms and developing diagnostic devices. In this study, we aim to optimize the activity, dynamic range, response time of one such biosensor: glucose dehydrogenase and calmodulin chimera (GDH-CaM). GDH-CaM functions as a universal core switch that can be utilized in designing biosensors for variety of analytes from small molecules to proteins1,2. Current response time of GDH-CaM biosensor is in the order of minutes, however for rapid diagnostic devices and therapeutic monitoring in clinics it is necessary to reduce the response time to seconds. For this purpose, we have developed a high-throughput analysis workflow for screening large domain insertion libraries. This workflow that combines rapid DNA amplification by colony RCA, Leishmania tarentolae cell-free expression and enzymatic activity analysis3. When this workflow was tested for GDH-CaM-403S biosensor as a control, the cell-free expressed GDH-CaM protein had highly similar reaction kinetics compared to the conventional E. coli protein expression and purification methods. Hence, we will now use this workflow to screen a large GDH-CaM domain insertion library covering 221 different insertion sites on GDH. The screening of a larger library of GDH-CaM chimeras with different insertion sites can allow us to correlate the location of the insertion site with the activation of the allosteric switch. This would enable a deeper understanding of GDH-CaM biosensor activation mechanism and kinetics.

  1. Guo Z, Murphy L, Stein V, Johnston WA, Alcala-Perez S, Alexandrov K. Engineered PQQ-Glucose Dehydrogenase as a Universal Biosensor Platform. J Am Chem Soc 2016; 138: 10108–10111.
  2. Guo Z, Johnston WA, Whitfield J, Walden P, Cui Z, Wijker E et al. Generalizable Protein Biosensors Based on Synthetic Switch Modules. J Am Chem Soc 2019; 141: 8128–8135.
  3. Kovtun O, Mureev S, Jung WR, Kubala MH, Johnston W, Alexandrov K. Leishmania cell-free protein expression system. Methods 2011; 55: 58–64.