An evaluation of genetically encoded FRET-based biosensors for quantitative metabolite analyses in vivo.

A broad range of genetically-encoded fluorescence biosensors has been developed, allowing the detection of signaling intermediates and metabolites in real time. Many of these biosensors are based on Foerster Resonance Energy Transfer (FRET). The two biosensors of the well-known "Venus-flytrap" type exemplarily studied in this work are composed of a central sugar binding protein flanked by two green fluorescent protein derivatives, namely ECFP as well as Citrine and EYFP, respectively. In order to evaluate FRET-based biosensors as an in vivo tool for quantitative metabolite analyses, we have thoroughly studied the effects of pH, buffer salts, ionic strength, temperature and several intracellular metabolites on the signal intensity of both biosensors and both fluorescence proteins. Almost all micro-environmental variations led to considerably different FRET signals, because either the fluorescent proteins or the metabolite binding domains were affected by the tested parameters. This resulted not only in altered FRET ratios between the apo state and the saturated state but also in significant shifts of the apparent binding constant. This underlines the necessity of careful controls in order to allow reliable quantitative measurements in vivo.

Application of immobilized bovine enterokinase in repetitive fusion protein cleavage for the production of mucin 1.

Bovine enterokinase is a serine protease that catalyzes the hydrolysis of peptide bonds and plays a key role in mammalian metabolism. Because of its high specificity towards the amino acid sequence (Asp)(4)-Lys, enterokinase is a potential tool for the cleavage of fusion proteins, which are gaining more importance in biopharmaceutical production. A candidate for adaptive cancer immunotherapy is mucin 1, which is produced recombinantly as a fusion protein in CHO cells. Here, we present the first repetitive application of immobilized enterokinase for the cleavage of the mucin fusion protein. The immobilization enables a facile biocatalytic process due to simplified separation of the biocatalyst and the target protein. Immobilized enterokinase was applied in a maximum of 18 repetitive reactions. The enzyme utilization (total turnover number) was increased significantly 419-fold compared to unbound enzyme by both immobilization and optimization of process conditions. Slight enzyme inactivation throughout the reaction cycles was observed, but was compensated by adjusting the process time accordingly. Thus, complete fusion protein cleavage was achieved. Furthermore, we obtained isolated mucin 1 with a purity of more than 90% by applying a simple and efficient purification process. The presented results demonstrate enterokinase to be an attractive tool for fusion protein cleavage.

Immobilisation of bovine enterokinase and application of the immobilised enzyme in fusion protein cleavage.

Two immobilisation methods for enterokinase were developed, which yielded high remaining activities for the cleavage of the fusion protein MUC1-IgG Fc. Different carrier materials were compared regarding remaining enzyme activity and storage stability. Immobilisation procedures involving support material activation using glutardialdehyde were found to result in low remaining activities. Applying less aggressive activation procedures, remaining activities of approximately 60% were received when immobilising enterokinase on either Estapor paramagnetic microspheres or hexamethylamino Sepabeads. In case of hexamethylamino Sepabeads we were able to increase the half-life time 4.3-fold at 23 degrees C and 3.8-fold at 4 degrees C compared to the free enzyme at the same temperatures. By immobilising the biocatalyst the downstream process is simplified allowing the easy removal of the enzyme from the reaction mixture. The immobilised enterokinase cleaves the fusion protein MUC1-IgG Fc in at least two repeated batches, proving the efficiency of the immobilisation method and the reusability of the biocatalyst.