Project 2014

1. The bacterial flagellum – New perspectives for protein scaffolds

The bacterial flagellum is a complex nanomachine that serves as a propulsive organelle for many species. It consists of a basal body, a motor complex and a helical filament that rotates due to the proton-powered motor. The filament is built up from multiple copies of a protein called Flagellin, about 20.000 duplicates of the protein can assemble in a helical pattern. Analysis of crystal structures revealed that the Flagellin of S. typhimurium is composed of three different domains. Whereas two of these domains, i.e. The N- and C-terminus respectively, are highly conserved among different bacterial species, the third domain is variable and can be exchanged. This domain is also located to the outer surface and therefore constantly available.

Due to the high number of Flagellin copies and the location of the variable loop the flagellum is an interesting structure to build a protein scaffold. We aim to insert different proteins in this unfunctional loop of the flagellum because protein scaffolds like this often provide practical advantages including elevated stability and high production yield in microbial expression systems.

1.1 The Silver Surfer

In today’s industrial-dominated world environmental pollution has become one of the biggest problems of our time. In addition to dangerous chemicals and greenhouse-gases that cause global warming, harmful quantities of heavy metals accrue as waste-products of the industry and get into the environment. Apart from that fact all living organisms need ions of transition metals which are essential for their survival since they are a crucial component of different proteins. Although these ions are important in low concentrations, a higher dose can lead to intoxication. If these metal ions are present in excess amounts important components of the cell can be damaged due to the formation of free radicals which is why environmental pollution by heavy metals is a rising problem of today’s society and a high health risk for humans as well.
Over the course of evolution a lot of organisms have developed mechanisms against metal ion stress. Parts of these mechanisms can be used to control the production of different proteins. We will combine these parts with the production of a modified protein which can bind several metal ions and is part of the flagellum, a long filamentous structure on the surface of the bacterium, mainly used for locomotion. The ion binding part of the protein is taken from the usual baker’s yeast.
This system enables the bacterium to produce different metal ion catching proteins depending on which kind of metal is present in its surrounding. A major advantage of the ion fishing flagellum is the extracellular collection of these ions. Furthermore bacteria and their flagella are relatively simple to separate from their surrounding and therefore suitable to collect and harvest metal ions.

Such a flagellum is capable of collecting a huge amount of metal ions. Finally, we will use our ‘silver surfer’ bacterium to selectively harvest silver, copper and other metals from solutions. The high selectivity and affinity of our massive ion catching flagellum will enable us to efficiently separate and withdraw ions from the surrounding environment. Employing heavy metal ion binding proteins would allow the bioremediation of contaminated areas and could be a major step towards reducing the effects of environmental pollution. Besides this obvious benefit of decontaminating heavy metal-loaded habitats, such a system can also be used to catch valuable metal ions for further recycling.

1.2 The Tumor Terminator

Searching for new cancer treatment options is one of the most relevant points of research worth striving for. Nowadays many treatments are based on a combination of different strategies like radiation, surgery and chemotherapy to increase the chances against cancer although there are many negative side effects of these quite unspecific or inefficient methods for healthy cells and tissues. Therefore the therapy has to be highly-specific in order to keep the collateral damage as low as possible.
The development of antibody therapy against cancer cells is promising because of its higher specifity and binding affinity for their target but reactions with endogenous antigens and the immune system make antibodies not always the optimal choice.
Every tumor cell has its own circumstances of genesis and characteristics of development which is why the treatment has to be adapted to these unique criteria.

Lung cancer is one of the most common cancer types with a poor prognosis and derives from epithelial cells. Special about these kinds of tumor cells is the expression of a surface protein called EpCAM (Epithelial Cell Adhesion Molecule) which is a prominent tumor marker. Lung carcinoma cells over express this cell adhesion molecule on the whole surface whereby healthy epithelial cells express the glycoprotein just on the basolateral site making EpCAM the ideal target structure for new treatments.
Thus we intend to insert an EpCAM-specific DARPin (Designed Ankyrin Repeat Protein) into the non-essential loop of the flagellin for targeting the characteristic structure as alternative to antibody-based scaffolds.

DARPins are synthetic proteins which are able to bind antigens and are structural related to ankyrin proteins. These consist of highly conserved repetitive amino acid motifs and are involved in protein-protein interactions. Their small size and the high affinity for an antigene which is comparable to an antibody make DARPins optimal for targeting.

The insertion of an EpCAM-specific DARPin into the flagellum could create binding affinity for tumor cells by induced proximity and also facilitate their elimination after binding by engineering cytotoxic ligands into the flagellum. In this manner the ratio between binding and effector components can be adjusted easily enabling the synthesis of mixed filaments.

Combining high-affinity DARPins for specific tumor cell targeting with a high concentration of coupled cytotoxic proteins could make the flagellum - a protein scaffold with homing and effector function at the same time - a new therapeutic approach for the fight against cancer without antibodies.

Team

We are an ambitious group of bachelor and master students from the fields of biology, biomedical science and chemistry joined together by our mutual interest in synthetic biology, boradening our horizon and having an awesome Team experience.