Having a library of Evobodies as well as a mutation system is a great way to guarantee a high variety of binding proteins. However, there is still the need of a screening method to separate Evobodies that show affinity to the target protein from those that do not. As we wanted our whole system to be an in vivo approach utilizing directed evolution we settled for three different approaches to screen for functional Evobodies:

The bacterial two-hybrid system, our main approach, consists of two fusion proteins and a reporter gene that grants selective advantage. One fusion protein is the target protein fused to a DNA-binding domain while the other comprises the Evobody together with a RNA polymerase subunit. The DNA-binding domain that is connected to the target protein binds upstream of the reporter gene, in our case coding for beta-lactamase. If there is a certain affinity between the Evobody and the target protein, the polymerase subunit recruits the full enzyme and subsequently the beta-lactamase is expressed. Cultivated in an increasing concentration of ampicillin, only those bacteria expressing enough beta-lactamase will prevail. Since the expression of beta-lactamase correlates to the affinity between Evobody and binding protein we can use the best grown bacteria to determine the sequence of the Evobody which then could be used for production.

The split-protein approach is also based on beta-lactamase as a resistance to ampicillin. The general idea is, to split a reporter protein into two non-functional subunits. Then, a translational fusion between one half of the split-protein respectively and one of the two allegedly binding proteins is created. In case of succesful binding between the proteins, the fused split-protein halfes should therefore restore the functional reporter protein. As afore mentioned we decided for beta-lactamase as a reporter since it allows for the application of selective pressure. Each, the Evobody and the target protein, are fused to a different half of the split-beta-lactamase. In case of a successful binding Evobody the bacterium in concern should be granted an ampicillin resistance due to a restored and functional beta-lactamase.

The TAT-hitchhiker selection works on a similar premise as the split-protein. But in this case the beta-lactamase is not divided into two subunits but instead its natural export signals are removed. this modified beta-lactamase forms a translational unit with the target protein whilst a signal peptide for the Tat (Twin-Arginine Translocation) pathway is attached to the Evobody. Only in case of a funtionally binding Evobody to the target protein is the beta-lactamase exported into the periplasm where it can counteract the ampicillin. Since the Tat pathway only exports correctly folded proteins, this approach also provides an option to check if the proteins are expressed as we hope they are.

In summary, we wanted a selection system to screen for Evobodies with high affinity to the target protein while being functional in vivo. We chose three approaches, the bacterial two-hybrid system, the split-protein approach and the TAT-hitchhiker selection.
References follow soon!