Grafting PNIPAAm from β-barrel shaped transmembrane nanoporesBio VI
Combining unique properties of transmembrane proteins (e.g., generating membrane potential gradients or translocation of compounds) with specific functions of polymers (e.g., temperature and pH stimuli handles) enables new applications. In order to assemble a protein – polymer conjugate, two strategies have been established: Using the grafting-to technique, pre-synthesized polymers are attached to the protein, whereas the grafting-from approach focusses on polymerizing monomers directly from a protein. Using globular proteins is well-established but investigation of membrane proteins for conjugate formation was not performed before. In this study, we established conjugate formations of a transmembrane protein using the β-barrel protein ferric hydroxamate uptake protein component A (FhuA), an outer membrane protein of Escherichia coli.
In vitro flow cytometry-based screening platform for cellulase engineeringBio VI
Screening technologies are of pivotal importance for tailoring biocatalysts in directed evolution, as millions of mutant enzyme variants could be generated in every trial. Hence, ultrahigh throughput screening techniques have been developed in order to still complete such trials in a reasonable amount of time. These techniques are well capable of analyzing up to 107 events per hour and thus can analyze the complete coverage of a generated protein sequence with high efficiency.
This technology becomes even more powerful if it is coupled with a cell-free enzyme expression technique. This expression method enables the experimentator to reduce diversity loss when transforming mutant libraries into expression hosts, to design enzymes of animal or human origin or even perform directed evolution of toxic enzymes.
The first ever combination of such a cell-free compartmentalization platform with a flow cytometry-based screening has been achieved in the InVitroFlow technology and successfully applied to directed evolution of cellulose enzymes.
Screening through the PLICable promoter toolbox enhances protein production in Escherichia coliBio VI
Escherichia coli is a common host for recombinant protein production in which product titers are highly dependent on the employed expression system. Thereby, promoters are a key element to control gene expression levels. In this study, a novel PLICable promoter toolbox was developed. It enables the identification of the most suitable promoter out of ten IPTG-inducible promoters (T7, A3, lpp, tac, pac, Sp6, lac, npr, trc and syn) for high level protein production in a single cloning step and after a screening experiment.