Wheat Germ Cell-Free Translation System as a Tool for In vitro Selection of Functional Proteins

摘要

We have demonstrated that mRNA, ribosome and resulting protein form complexes (ternary complexes) in wheat germ cell-free translation system and these complexes are stable for at least several hours. The protein folds into a proper conformation capable of specific binding with the inhibitor of its enzymatic activity. The removal of the stop codon from mRNA does not affect translation and mRNA-ribosome-protein complex stability. We have used these results to develop a method of isolation of mouse dihydrofolate reductase (mDHFR) encoding mRNA from native pool of mouse liver mRNA. The native pool of mouse liver mRNA was translated in vitro in a wheat germ cell-free translation system (WG-CFS), and enzyme-specific ternary complexes were affinity selected on a methotrexate-BSA coated 96-well microtiter plate (methotrexate, MTX, is an inhibitor of DHFR enzymatic activity). Bounded ternary complexes were eluted by MTX treatment. mRNA from eluates was amplified by template-switch RT-PCR and products of RT-PCR analyzed by gel electrophoresis. The cDNA was amplified by one-step reverse transcription-PCR and used for transcription, followed by translation and determination of the DHFR enzymatic activity in translation mixtures. This method is suitable for direct cDNA cloning from mRNA or cDNA libraries and for investigation of protein-protein interactions.