Figure 1

Preparation method of cDNA display. (A) Scheme of cDNA display preparation. The mRNA-linker conjugate was prepared by ligation of a puromycin-linker to the 3’-terminus of an mRNA coding B-domain of protein A (BDA). The mRNA-linker conjugate was translated by an in vitro translation reaction. The produced mRNA-protein fusion molecule and the remaining mRNA-linker conjugate were captured with SA-beads from the translation reaction mixture and reverse transcribed on the beads. The mRNA/cDNA-protein fusion molecule and the mRNA/cDNA molecule were released from the SA-beads by RNase T1 treatment. The mRNA/cDNA-protein fusion molecule was purified by the His-tag in the translated protein. (B) Schematic diagram of construct of short biotin segment puromycin-linker (SBP-linker). The SBP-linker construct comprises four parts: a ligation site for mRNA, a primer region for reverse transcription, a biotin moiety for the immobilization of the mRNA-linker conjugate on Streptavidin-beads, and two cleavage sites for RNase T1 to release the mRNA/cDNA-protein fusion molecule from the SA-beads. In addition, the SBP-linker includes puromycin (for the covalent linking of the expressed protein to mRNA) and fluorescein (for detection and quantification). The 3’-region of the mRNA is shown in lower case letters. [N-(6-maleimidcaproyloxy) succinimide] (EMCS) is bifunctional cross-linker used in the preparation of the SBP-linker.