(75g) Streamlined Protocol for mRNA Display

In vitro directed evolution techniques such as ribosome display and mRNA display are powerful tools for protein engineering, capable of handling libraries containing trillions of members. mRNA display uses an mRNA-DNA-puromycin fusion as template for in vitro translation, which results in a covalent puromycin linkage between the mRNA and the corresponding nascent peptide, thus creating a highly stable selection particle which is useful for protein/peptide stability selection experiments. Our objective in this study was to establish a streamlined protocol for mRNA display based on a ribosome display protocol currently used in our laboratory. The mRNA display protocol is significantly streamlined and optimized, which should enable faster and easier directed evolution experiments, thus improving the in vitro development of therapeutic proteins and peptides. Advancements to the protocol included: ultrafiltration rather than gel-purification to purify the ligation reaction between mRNA and DNA-puromycin; use of PURExpress in vitro protein synthesis kit (New England Biolabs) rather than a lysate-based system to maximize the number of functional selection particles; and optimization of the conditions used to generate and purify the selection particles. PURExpress offers better reproducibility, lack of nucleases, and a high concentration of ribosomes. Relative yield of mRNA-peptide was determined by quantitative RT-PCR following affinity selection. We were able to produce consistent results with selection particles displaying a peptide (3xFLAG) or a protein (off7, a designed ankyrin repeat protein). The crucial steps at which efficiency may typically suffer, including ligation of mRNA to the DNA-puromycin linker and covalent binding of puromycin to the nascent polypeptide chain, have been found to be adequately efficient in our system. Ongoing experiments in our laboratory are utilizing this streamlined method to select peptide binders from a naïve library.