<jats:sec><jats:label /><jats:p>Streptavidin is used widely in biotechnology because of the exceptional stability of its binding to biotin. This stability is, however, limiting for applications such as imaging and nanotechnology. We engineered a mutant streptavidin with much slower dissociation from biotin. This mutant streptavidin also had increased mechanical strength and thermostability. We applied this mutant to analyze force generation by FtsK, one of the fastest known molecular motors. FtsK displaced proteins bound in its path and induced rapidly the release of streptavidin from biotinylated DNA. On the other hand, the mutant streptavidin resisted displacement, indicating the force generated by FtsK translocation. The resilience of the binding of this mutant streptavidin may find many applications in biotechnology and biophysics. Funding was provided by the Wellcome Trust, the Biotechnology and Biological Sciences Research Council Chemical Biology DPhil programme and Somerville College Oxford.</jats:p></jats:sec>
