21ENGBIO - Peptide excision, replacement and ligation (PERL) as a new strategy for protein engineering

In this project, we will develop a completely new way to re-engineer the basic structure of proteins. Engineering of proteins is a widely-used approach for the production of biopharmaceuticals, for the study of processes in the living cell and for the synthesis of complex materials. Proteins are formed of long polymers of amino-acids which fold up to form complex three-dimensional shapes. The majority of changes that can currently be made are simple additions to the surface of these particles. To use an analogy, these are the molecular equivalent of repainting a car or adding new tire rims. Larger changes to the shape and structure of the protein can be made by editing the protein before it is first made (effectively before it leaves the factory) but ultimately these modifications must be made of the same materials as the original protein and so some modifications are simply inaccessible at the moment.

What it is not currently possible to do is to make large modifications to the protein once it has been made; to remove an element of the finished, folded protein and replace it with a new part while retaining a functional protein. In this project, we are seeking to do just that: we will develop methods to carry out the molecular equivalent of turning a car into a convertible or switching out the engine! Based on some recently developed technology in our laboratory to rejoin the protein parts, we will develop a method that will ultimately allow us to remove a part of the protein and replace it with alternative parts made from different materials that cannot be inserted using the machinery of the cell.

We will define a new strategy for protein engineering in which portions of the peptide backbone are removed and replaced with synthetic fragments. This approach is depending upon sequential addition of a range of optimised protein-modifying enzymes. During this short project, we will demonstrate the first application of this strategy to folded proteins using a range of model proteins varying in size between 10 and 100 kDa and demonstrate insertion of a range of functional synthetic inserts. This project will provide essential proof-of-concept for a technology that will then be applicable across a wide range of applications in protein chemistry, cellular biology and biopharmaceutical development.