Question

You are a protein engineer employing yeast display directed evolution to engineer novel proteins that bind with high affinity and specificity to a target of interest. You' ve developed a peptide that binds to the extracellular membranous target, Evolutionary Growth Factor Receptor (EmGF - R).
You developed a binder in vitro using yeast display screened against soluble EmGF -  R. Unfortunately, when the binder was tested in vivo, it performed poorly.
You hypothesize the binding epitope is hidden in an intramembraneous domain when EmGF - R is in its native cellular position.
Develop a new set of directed evolution experiments to ensure you develop a binder against an extracellular epitope.

Answer 1

Few points can be considered while designing experiments in order to develop a binder against an extracellular epitope:

1. Use of scaffold proteins and enzymes have been used in order to increase binding, activity, and specificity.

2. Signaling components are tether or together by the use of scaffold protein in order to increase efficiency.

3. Components are localized to the specific targets protein.

4. Scaffold protein helps in protection or insulating components from inactivation or degration.

5. Another advantage of using scaffold protein is that negative and possitive feedback are well co-ordinated.