A High Performance Platform Based on cDNA Display for Efficient Synthesis of Protein Fusions and Accelerated Directed Evolution

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• 作者：Mohammed Naimuddin ; Tai Kubo
• 刊名：ACS Combinatorial Science
• 出版年：2016
• 出版时间：February 8, 2016
• 年：2016
• 卷：18
• 期：2
• 页码：117-129
• 全文大小：661K
• ISSN：2156-8944

文摘

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.