Description

Introduction

Our project is called SPACE-P (Structural Phage Assisted Continuous Evolution of Proteins) and is part of the Synthetic Biology field. It focuses on accelerating the process of developing protein binding partners, which is required in pharmaceutical research as well as biotechnology and many other fields of science. Thus far phage display is the most commonly used method for the discovery of protein binding partners. In this method a large library of potential binding partners is created where the strongest candidates are selected for further affinity testing. This process is very time consuming, cost intensive, requires human intervention steps, and is limited to the size of the given library. Our platform will make it possible to start with a single molecule, transform it by mutation and selection pressure to improve the binding affinity towards a target protein. This method is not only easy to implement, utilizing simple organisms and devices, but also significantly faster and cheaper than currently used tools.

Subprojects

To establish our idea we divided the whole work into subprojects which were realized simultaniously. These subprojects should be combined later. The error finding and erasing is much easier with this approach and it is faster compared to a step by step approach where you try to start with the next step only when you succeeded the previous ones. The subprojects we worked on are the following:

Correct folding study of target protein

As a proof of principle we wanted to fit an affibody to the epitope of a target protein. The target we chose for our studies is a human membrane protein called human epidermal growth factor receptor 2 (HER2). To use a human protein and membrane protein is challenging since the codon usage, post-translational modifications, disulfide bridges and membrane parts have to be taken into account. To deal with this challenges we decided for a small extracellular domain that carries no post-translational modifications and disulfide bridges. This part of HER2 is involved in ligand binding and therefore is an interesting drug target. Afterwards we harmonized the codon usage to E. coli. Finally we compared the protein structure expressed from E. coli to the expected one from literature. To do so we cloned the fragment into an expression vector with a tag, purified it with a column and analyse it with circular dichroism spectroscopy.

Structure analysis of our targets and their interactions

Having the crystalized structure of the extracellular part of HER2 and the affibody ZHER2 binding to it (PDB-ID: 3MZW), we are able to perform further investigations on our targets and their interactions. Those analysis should give us a presentiment of possibilities for the directed evolution and therefore provide a benchmark for the choice of further possible targets. The following analysis are performed:

• Calculation of intefacial residues of HER2 and its bound affibody
• Calculation of electrostatic interactions in the interface
• Conservation study of HER2
• Visualization of the b-factor for the affibody ZHER2

Figure 1 - HER2 conservation - calculated using 11 HER2 structures from different organisms.

The following video shows the structure of the extracellular regions of HER2 with the affinity matured 3-helix affibody ZHER2 (PDB-ID: 3MZW) and focuses on their interaction, whereas hydrogen bonds are represented as dashed yellow lines and then the complete interacting interface is represented as surface, colored by atom type (N-blue, O-red).

Affibody ZHER2 surface coloured by b-factor	Affibody ZHER2 structure coloured by b-factor	Affibody ZHER2 structure coloured by b-factor

Investigation of P3 threshold for E. coli resistance

The protein III (P3) from M13 plays a key role in our concept. The deficiency of it in the M13 phage we used can be compensated by the modified E. coli. However it is known that the expression of P3 in E. coli before the infection with M13 leads to the resistance of the bacterium to the phage. This was our motivation to find the threshold of resistance. To find it we measured the amount of infected bacteria cells in the lagoon depending on the expression rate of P3. The P3 expression rate should be detected by the coexpression of a fluorescent protein while the amount of infected cells was conducted with the help of a blue-white screening. In this assay the cells carrying a phage are blue on X-Gal plates due to the complementation of the lacZ gene by the phages.

Conversion of BACTH into an iGEM standard and analysis of function

Set up of flow system