cDNA Display of Disulfide-Containing Peptide Library and In Vitro Evolution.

Directed in vitro evolution (IVE) is now a widely applied technology to obtain molecules that have designed new features of one's demands. We describe here experimental procedures for a cDNA display IVE to select peptide aptamers from a scaffold-based random peptide library. A three-finger (3-F) peptide library is exemplified, which has been shown its pluripotency to various target molecules. Peptide scaffolds including 3-F are refined through evolution, and they are mostly stabilized by disulfide bridges. To utilize such disulfide-containing protein library in IVE, we optimized the translation and folding conditions. Co-translational folding assisted by protein disulfide isomerase was found to have better efficiency than posttranslational refolding in the IVE. Linker is also a key element to make a tight genotype-phenotype linkage. Here, we introduced a whole procedure of IVE to use a newly designed puromycin linker, which was synthesized by a novel branching strategy. The improved linker enabled rapid and highly efficient ligation of mRNA and synthesis of protein fusions.