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Project

Abstract

Protein-protein interactions play a key role in biology. Designing and coordinating interactions in order to discover new drugs comes with a host of challenges. Our goal is to modify phage-assisted continuous evolution (PACE), specifically for protein interactions. PACE combines the bacteriophage M13 and Escherichia coli in a dynamic scheme whereby M13 only survives if it infects E. coli. This is achieved when the viral protein P3 is expressed. SPACE-P aims to incorporate a key-lock mechanism that regulates the expression of P3. In our model, the interaction between the protein HER2 and an affibody will be the key to open the lock. Over several phage life cycles, evolutionary pressure will favour the interactions with the greatest yield of P3, thereby increasing that phages virulence and the continued evolution of that particular affibody. Our method will reduce the time and cost of drug discovery and enable the interaction between many choose-able proteins.

Do you want to know more about our project?

We have subdivided the Project section in six different parts:

• Description
• Background
• Methods
• Results
• Parts
• Conclusions

Overview

What is SPACE-P?

- Combining three interesting technologies to make way for an innovative idea is what SPACE-P is all about! We'll be exploring the space within bacteria to carry-out our madness! SPACE-P stands for Structural Phage Assisted Continuous Evolution of Proteins.

What is SPACE-P?

- Combining three interesting technologies to make way for an innovative idea is what SPACE-P is all about! We'll be exploring the space within bacteria to carry-out our madness! SPACE-P stands for Structural Phage Assisted Continuous Evolution of Proteins.

Okay, why do we need SPACE-P?

- Our goal is to speed up screening for potential peptide sequences of disease or target proteins that are capable of interacting with antibodies.

But how does SPACE-P translate to your goal?

- We try to evolve an affibody molecule to fit better with the protein of interest. In view of screening for potential peptides or proteins we also want to validate the binding of the affibody ZHER2 with the protein HER2 (Human epidermal growth factor receptor), to present a new way to identify potential drug epitopes. You can read more about affibodies here.

So how do you plan to do it?

- We combine the 3 different techniques of "Phage Display", "BACterial Two Hybrid (BACTH) system", and "Phage assisted continuous Evolution" to evolve the affibody that fits the target protein. We use a lock and key model to evolve the affibody protein.

How does this process work?

- We try to evolve the antibody which is encoded in the M13's phage genome. The target protein is encoded in the E. coli genome. Now, naturally the M13 would try to infect the E. coli to replicate. Here's the catch, we use the BACTH system as a lock and key model on the E. coli strain and the M13 phage DNA.

The lock in question is a 3-step lock system that is designed in such a way that:

1. The 2 domains of the enzyme adenylate kinase are split, one subsequence is bound to the affibody and is located in the M13 phage genome, and the other is in an accessory plasmid in the E. coli system and connected to the target protein. These two domains have to interact to be able to produce cAMP.
2. The protein P3 that is essential for phage infectivity is deleted in the M13 bateriophage.
3. Cool down a cooling centrifuge to 2 °C.