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Updated: Jan 21, 2026
How to Stabilize Protein: Stability Screens for Thermal Shift Assays and Nano Differential Scanning Fluorimetry in the Virus-X Project
Published on: February 11, 2019
Nanobody stability engineering by employing the ΔTm shift; a comparison with apparent rate constants of heat-induced
Patrick Kunz1, Aurelio Ortale1, Norbert Mücke2
1Division of Functional Genome Analysis, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, Heidelberg, Germany.
The antigen-binding domains of camelid heavy-chain antibodies, also called nanobodies, gained strong attention because of their unique functional and biophysical properties. They gave rise to an entire spectrum of applications in biotechnology, research and medicine. Despite several reports about reversibly refolding nanobodies, protein aggregation plays a major role in nanobody thermoresistance, asking for strategies to engineer their refolding behavior. Here, we use measurements of nanobody aggregation kinetics to validate structural features in the nanobody fold that are suppressing heat-induced nanobody aggregation. Furthermore, the kinetic measurements yielded a detailed insight into the concept of the ΔTm shift, a metric for protein aggregation propensities obtained from differential scanning fluorimetry measurements. By relating the equilibrium measurements of the ΔTm shift to the kinetic measurements of heat-induced nanobody aggregation, a distinct relationship could be identified that allows a prediction of nanobody aggregation rates from a simple equilibrium measurement of ΔTm.
The antigen-binding domains of camelid heavy-chain antibodies, also called nanobodies, gained strong attention because of their unique functional and biophysical properties. They gave rise to an entire spectrum of applications in biotechnology, research and medicine. Despite several reports about reversibly refolding nanobodies, protein aggregation plays a major role in nanobody thermoresistance, asking for strategies to engineer their refolding behavior. Here, we use measurements of nanobody aggregation kinetics to validate structural features in the nanobody fold that are suppressing heat-induced nanobody aggregation. Furthermore, the kinetic measurements yielded a detailed insight into the concept of the ΔTm shift, a metric for protein aggregation propensities obtained from differential scanning fluorimetry measurements. By relating the equilibrium measurements of the ΔTm shift to the kinetic measurements of heat-induced nanobody aggregation, a distinct relationship could be identified that allows a prediction of nanobody aggregation rates from a simple equilibrium measurement of ΔTm.
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