Team:TU Dresden/Human Practices/Long/Translation Poster

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Poster for the LNdW

English translation

Motivation

Protein-protein interactions are a common principle for the functionality of medical drugs, such as monoclonal antibodies in cancer therapy. The fabrication of those active agents is very time and cost intensive. With our project we propose an alternative way, the use of one of the smallest antigen binding molecules currently known, the affibody, instead of a monoclonal antibody.

Identification and evolution of a binding partner/ligand is often performed through phage display. To identify binding partners, this technique accesses a limited library of proteins, performs time consuming in vitro evolution and selection, in order to hopefully find a strong interaction.

In SPACE-P we want to overcome these timely limitations with a targeted and continuous method, which is based on the use of bacteriophage M13.

Functional principle

The information of protein P3 (1) is destroyed in the genome of our phage M13. Nevertheless, P3 is essential for the proliferation of the phages (2). Only if the affibody (3) adapts to the structure of our target protein (4) P3 is produced by the host. This creates evolutionary pressure for a binding interaction to develop over the course of a few days. The easement to this artificially generated pressure is a process that includes several steps. If the affibody mutates in a way that it fits to the target protein, a split enzyme (T18/T25) is reunified. Consequently, it regains its functionality, the conversion of ATP into cAMP (6). Subsequently cAMP activates a lacZ promotor that regulates P3 production (7). Therefore, favorable mutations result in a better adaptation of the protein to its target (8).

The continuous evolution and selection takes place in a lagoon, where unfavorably mutated phages are washed out and favorably mutated phages become concentrated, remaining infectious towards the E. coli host, also continually flowing in the lagoon.

Experiment setup

In order to constrain evolution to only the phages, the flow rate is optimized such that E. coli host cells are maintained in at their exponential growing phase, before proliferation. This is performed with a chemostat which continuously supplies plain growing media and extracts E. coli rich media. Since only a small portion of bacteria is required, one portion of the media rich with bacteria is discarded and the other portion goes to the lagoon, where they meet the phages and where evolution can take place.