AbstractsBiology & Animal Science

Genetic selection systems to study and optimize protein folding in vivo

by Antje Schickert

Institution: Universität Tübingen
Year: 2015
Record ID: 1112839
Full text PDF: http://hdl.handle.net/10900/60511


Most proteins have to fold into their native structure, a distinct three-dimensional fold, in order to perform their functions in the cell. Many proteins can reach their native state without the help of other macromolecules in vitro, but the crowded environment of the cell constitutes a more challenging folding environment. Biosensors can facilitate studying protein folding in the cell by linking protein stability of a guest protein to antibiotic resistance. Biosensors designed in our lab consist of an antibiotic resistance marker with a guest protein inserted into a permissive site. The folding status of the guest protein can be easily assessed by measuring the antibiotic resistance of cells expressing these biosensors. This method allowed us to investigate the folding pathway of bovine pancreatic trypsin inhibitor (BPTI) directly in the complex environment of the cell. The biosensor proved especially powerful when we selected for protein variants that increased protein expression by demanding growth on high antibiotic concentrations. Most BPTI variants that achieved increased expression did so by eliminating or destabilizing the disulfide bond Cys14-Cys38 in BPTI. This had the effect of reducing the formation of kinetically trapped intermediates during folding, which are more susceptible to aggravation or proteolysis. Previously the lab had developed biosensors that only worked in the periplasm of prokaryotes. I developed a biosensor that could function in the cytosolic compartment. While doing so I established a protocol that allows one to easily identify permissive sites in antibiotic resistance proteins. This approach gave rise to three new cytosolic biosensors, all of which exhibited a linear correlation between antibiotic resistance and protein stability in vivo for different guest proteins independently of the exact position of the permissive site in the resistance marker. The expansion of our antibiotic resistant biosensors into the cytosolic compartment offers the opportunity to investigate the effect of chaperones on proteins directly in the cell. The stabilizing effect of polyphosphate was measured using the antibiotic resistance proteins, which provided in vivo evidence for polyphosphates chaperone activity. Biosensors are efficient tools for investigating proteostasis in vivo. Using this method we can assess the influence of cellular components such as chaperones on protein folding and expression, and monitor the formation of amyloids. Furthermore, the biosensors allow us to evolve guest proteins in order to improve their folding properties simply by selecting for increased antibiotic resistance. This makes these biosensors effective tools for optimizing the expression of problematic biotechnological or pharmaceutically interesting proteins.; Um ihre Funktion in der Zelle ausüben zu können, müssen viele Proteine sich in eine spezifische dreidimensionale Struktur, ihre native Form, falten. In vitro können viele Proteine ohne die Unterstützung andere Makromoleküle falten, aber im hoch…