| Popis: |
More than a thousand different protein folds are known to exist, many of which have been characterized through dedicated research; however, there is little evidence of how these numerous structural forms arose through evolution. One approach to elucidating possible evolutionary pathways of protein structure is to perform directed evolution experiments on ancestrally related protein families which occupy different folds. One approach to the directed evolution of a protein fold is to follow a destroy-and-rebuild scheme where protein stability is knocked out through protein engineering, random mutagenesis is used to resurrect stability, and stable variants are selected through the powerful method of phage display coupled to proteolysis. The Cordes lab has previously identified a case suitable for such an investigation in the Cro protein family where two small DNA binding proteins, Xfaso 1 and P 6, occupy different folds yet retain high sequence identity. Previous work has identified deletion mutations which destroy the stability of the ancestral α-helical fold, but are also known to be compatible with the descendant β-sheet fold. Here, error prone PCR is presented as a possible method of resurrecting stability through random mutagenesis in Cro protein variants destabilized through these deletions. A protocol has been optimized for the production of mutant libraries of these genes Through EP-PCR. Test clones of these libraries into a prototype pCANTAB B phagemid vector for later phage display investigations have been successful, and sequencing has revealed that the implemented protocol yields an acceptable nucleotide substitution rate of 2.9%. |
