Final Report Summary - EXIVC (Expanding the Potential of in vitro Compartmentalised Screening and Selection Approaches) The PURE system (Protein Synthesis Using Recombinant Elements) was adapted for selection of binding proteins using SNAP-tag display was adapted for SNAP-tag display of antibody fragments in selections for protein binders to lysozyme (as a model target protein) . The SNAP-tag technology had been developed in the Cambridge group and consists of the in vitro creation of a covalent genotype-phenotype linkage between DNA and expressed protein in microdroplets that ensure that the very protein encoded by a linear DNA fragment is covalently coupled to it. Recent work of the Hollfelder group had shown that the protein expression efficiency in droplets in limited and may significantly impede progress in directed evolution indroplets. Therefore I probed whether these issues could be addressed in a different formulation, adding DNAK, GroEL and protein disulfide isomerases (PDIs). These measures have been shown to improve the expression of antibodies, for which selections have to be carried out under oxidative conditions. To this end I have performed model selections that yielded antibodies with μM binding affinity. Controls suggested that successful selections were only possible using the newly developed protocol: In the presence of 3.5 mM of glutathione, the addition of PDI improved recovery by >30-fold. Western blotting showed that antibody binding is not compromised under these conditions.Building in this procedure I wanted to conduct selections with carbohydrate binding domains (CBDs) and started to clone a selection of these proteins. A number of these proteins did not show good expression in in vitro expression systems, neither in PURE, nor in commercial Roche or Invitrogen kits. Towards the end of the project I have established that visible enrichment can be observed in model selections with one CBD. Proper selections could not be performed in the timeframe of the project, because of the many practical obstacles encountered. However, other members of the host group will take up this project in the future. The – by now robust – protocol to perform selections from protein libraries with disulfide bonds will help to achieve the goal of actual selection of CBDs. I have set up assays for catalytic selections that will allow screening of a catalytically competent protein after evolving the binding capabilities of the CBD. Documents connexes final1-220701-742191-ohashi-final-report.pdf
