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Identifying mutations that stabilize proteins is challenging because most substitutions are destabilizing. In addition to being of immense practical utility, the ability to evolve protein stability in vivo may indicate how evolution has formed today's protein sequences. Here we describe a genetic selection that directly links the in vivo stability of(More)
To optimize the in vivo folding of proteins, we linked protein stability to antibiotic resistance, thereby forcing bacteria to effectively fold and stabilize proteins. When we challenged Escherichia coli to stabilize a very unstable periplasmic protein, it massively overproduced a periplasmic protein called Spy, which increases the steady-state levels of a(More)
Conditionally disordered proteins can alternate between highly ordered and less ordered configurations under physiological conditions. Whereas protein function is often associated with the ordered conformation, for some of these conditionally unstructured proteins, the opposite applies: Their activation is associated with their unfolding. An example is the(More)
We have devised protein-folding sensors that link protein stability to TEM-1 β-lactamase activity. The addition of osmolytes and other compounds with chemical chaperone activity to the growth medium of bacteria containing these sensors increases β-lactamase activity up to 207-fold in a dose-dependent manner. This enables the rapid detection and sensitive(More)
The periplasm provides a strongly oxidizing environment; however, periplasmic expression of proteins with disulfide bonds is often inefficient. Here, we used two different tripartite fusion systems to perform in vivo selections for mutants of the model protein bovine pancreatic trypsin inhibitor (BPTI) with the aim of enhancing its expression in Escherichia(More)
HdeA is a periplasmic chaperone that is rapidly activated upon shifting the pH to acidic conditions. This activation is thought to involve monomerization of HdeA. There is evidence that monomerization and partial unfolding allow the chaperone to bind to proteins denatured by low pH, thereby protecting them from aggregation. We analyzed the acid-induced(More)
We describe here a genetic selection system that directly links protein stability to antibiotic resistance, allowing one to directly select for mutations that stabilize proteins in vivo. Our technique is based on a tripartite fusion in which the protein to be stabilized is inserted into the middle of the reporter protein β-lactamase via a flexible linker.(More)
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