PURE mRNA display and cDNA display provide rapid detection of core epitope motif via high-throughput sequencing.

The reconstructed in vitro translation system known as the PURE system has been used in a variety of cell-free experiments such as the expression of native and de novo proteins as well as various display methods to select for functional polypeptides. We developed a refined PURE-based display method for the preparation of stable messenger RNA (mRNA) and complementary DNA (cDNA)-peptide conjugates and validated its utility for in vitro selection. Our conjugate formation efficiency exceeded 40%, followed by gel purification to allow minimum carry-over of components from the translation system to the downstream assay enabling clean and efficient random peptide sequence screening. We chose the commercially available anti-FLAG M2 antibody as a target molecule for validation. Starting from approximately 1.7 × 1012 random sequences, a round-by-round high-throughput sequencing showed clear enrichment of the FLAG epitope DYKDDD as well as revealing consensus FLAG epitope motif DYK(D/L/N)(L/Y/D/N/F)D. Enrichment of core FLAG motifs lacking one of the four key residues (DYKxxD) indicates that Tyr (Y) and Lys (K) appear as the two key residues essential for binding. Furthermore, the comparison between mRNA display and cDNA display method resulted in overall similar performance with slightly higher enrichment for mRNA display. We also show that gel purification steps in the refined PURE-based display method improve conjugate formation efficiency and enhance the enrichment rate of FLAG epitope motifs in later rounds of selection especially for mRNA display. Overall, the generalized procedure and consistent performance of two different display methods achieved by the commercially available PURE system will be useful for future studies to explore the sequence and functional space of diverse polypeptides.

Phospholipid synthesis inside phospholipid membrane vesicles.

Construction of living artificial cells from genes and molecules can expand our understanding of life system and establish a new aspect of bioengineering. However, growth and division of cell membrane that are basis of cell proliferation are still difficult to reconstruct because a high-yielding phospholipid synthesis system has not been established. Here, we developed a cell-free phospholipid synthesis system that combines fatty acid synthesis and cell-free gene expression system synthesizing acyltransferases. The synthesized fatty acids were sequentially converted into phosphatidic acids by the cell-free synthesized acyltransferases. Because the system can avoid the accumulation of intermediates inhibiting lipid synthesis, sub-millimolar phospholipids could be synthesized within a single reaction mixture. We also performed phospholipid synthesis inside phospholipid membrane vesicles, which encapsulated all the components, and showed the phospholipids localized onto the mother membrane. Our approach would be a platform for the construction of self-reproducing artificial cells since the membrane can grow sustainably.