title: A High Performance Platform Based on cDNA Display for Efficient Synthesis of Protein Fusions and Accelerated Directed Evolution.
abstract: We describe a high performance platform based on cDNA display technology by developing a new modified puromycin linker-oligonucleotide. The linker consists of four major characteristics: a "ligation site" for hybridization and ligation of mRNA by T4 RNA ligase, a "puromycin arm" for covalent linkage of the protein, a "polyadenosine site" for a longer puromycin arm and purification of protein fusions (optional) using oligo-dT matrices, and a "reverse transcription site" for the formation of stable cDNA protein fusions whose cDNA is covalently linked to its encoded protein. The linker was synthesized by a novel branching strategy and provided >8-fold higher yield than previous linkers. This linker enables rapid and highly efficient ligation of mRNA (>90%) and synthesis of protein fusions (∼ 50-95%) in various cell-free expression systems. Overall, this new cDNA display method provides 10-200 fold higher end-usage fusions than previous methods and benefits higher diversity libraries crucial for directed protein/peptide evolution. With the increased efficiency, this system was able to reduce the time for one selection cycle to <8 h and is potentially amenable to high-throughput systems. We demonstrate the efficiency of this system for higher throughput selections of various biomolecular interactions and achieved 30-40-fold enrichment per selection cycle. Furthermore, a 4-fold higher enrichment of Flag-tag was obtained from a doped mixture compared with that of the previous cDNA display method. A three-finger protein library was evolved to isolate superior nanomolar range binding candidates for vascular endothelial growth factor. This method is expected to provide a beneficial impact to accelerated drug discovery and proteome analysis.
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology , Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.|Janusys Corporation , #508, Saitama Industrial Technology Center, Skip City, 3-12-18 Kami-Aoki, Kawaguchi, Saitama 333-0844, Japan.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Naimuddin
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology , Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.|Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST) , 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan.
authors.forename: Tai
authors.initials: T
authors.lastname: Kubo
mesh_terms: D018076:DNA, Complementary / Q000737:chemistry*; D019020:Directed Molecular Evolution / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry; D019151:Peptide Library; D010455:Peptides; D011487:Protein Conformation; D011691:Puromycin / Q000737:chemistry; D011130:RNA Ligase (ATP); D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D014764:Viral Proteins
chemical_list: D018076:DNA, Complementary; D009841:Oligonucleotides; D019151:Peptide Library; D010455:Peptides; D011994:Recombinant Proteins; D014764:Viral Proteins; D011691:Puromycin; D011130:RNA Ligase (ATP); C467724:bacteriophage T4 RNA ligase 2
keywords: cDNA display; directed evolution; drug discovery; high throughput; protein fusions; puromycin linker