Figure 1

Overview of cDNA display and construction of the three-finger (3F) scaffold library. (A) cDNA display is based on the formation of a covalent fusion between the expressed protein (phenotype) and the encoding cDNA (genotype) via a puromycin attached to an oligonucleotide linker. The puromycin linker is central to this technology (see B). In step 1, the library is prepared with the construct to be adopted for the cDNA display (Additional file 1, Fig. S1 and Additional file 2, Table S1 online), transcribed into mRNA and ligated to the puromycin linker in the presence of T4 RNA ligase (steps 2 and 3). The mRNA-linker conjugate is then translated in a cell-free translation system (reticulocyte lysate) and the covalent linkage between the linker and protein is formed in the presence of high salt (step 4). Rapid purification is achieved using biotin-streptavidin. cDNA is then synthesized by reverse transcription in step 5 utilizing the 'built-in' primer of the linker. In step 6, the genotype-phenotype complex is released from the beads by restriction digestion with Pvu II. Further, full length proteins are purified via the C-terminal 6xHis (step 7). Following selection of molecules against the immobilized targets, the bound molecules are recovered, purified, amplified (steps 8-10) and used in the next round of selection. (B) Schematic of the Puromycin linker and its functional features. The puromycin linker used in this study consists of four essential functional features. First, a ligation site for mRNA, a biotin moiety for rapid purification using immobilized streptavidin (SA), a primer region for reverse transcription and a restriction site for the release of the complex from the immobilized SA beads.