Research area
Reconstituted cell-free systems (POPFLEX and i-POPFLEX)
High-performance cell-free protein synthesis has transformative potential for synthetic biology, yet the prohibitive costs of PURE kits and the labor intensity of in-house preparation have restricted accessibility and scalability. We developed i-POPFLEX (Purified components OPtimized for FLEXible protein expression), a modular cell-free protein synthesis system in which 34 translation factors and a split T7 RNA polymerase are individually synthesized in vitro and assembled using automated liquid handling. This workflow minimizes manual input and supports parallelized production, generating complete, ready-to-use systems within two days. i-POPFLEX achieves up to 8.4-fold higher protein yields and a 96 % cost reduction (27-fold lower cost) compared with commercial kits. Its flexible architecture also enables selective component inclusion for genetic code reprogramming and site-specific incorporation of non-canonical amino acids. By coupling modular design with automation, i-POPFLEX provides an accessible, customizable, and economically viable platform for next-generation biomanufacturing workflows.
Expanding ribosomal synthesis of non-standard cyclic backbones in vitro
Cyclic motifs, found in diverse natural products such as penicillin V, goadsporin, and patellamide C, are widely recognized for their exceptional pharmacological efficacy. Traditionally, these cyclic structures have been produced through indirect methods, including enzymatic post-translational modifications, where a linear peptide is first synthesized by the ribosome and then cyclized by specific enzymes or solid-phase peptide synthesis (SPPS). However, these approaches often face limitations in terms of productivity, overall efficiency, and the scalability required for constructing diverse, high-throughput libraries. To address these challenges, our research focuses on the ribosome’s Peptidyl Transferase Center (PTC), a chemical nexus that facilitates peptide bond formation through a unique mechanism known as "entropy trapping". By introducing bifunctional non-canonical monomers (ncMs) into this specialized environment, we harness the ribosome's substrate constraining ability to drive successive ring closing reactions in situ. This process enables the direct synthesis of robust cyclic motifs within the ribosomal framework. Furthermore, we conduct in-depth investigations into the thermodynamic and kinetic principles governing these ribosome-mediated reactions to establish fundamental rules for ring closing reactions. These mechanistic insights serve as a blueprint for designing ncMs that allow for sequence-defined and site-specific incorporation into diverse chemical motifs. By transforming the ribosome into a versatile synthesis platform, we aim to revolutionize the discovery and production of next-generation medicinal molecules.
Developing Novel Peptide Drugs by Diverse ncAAs
Peptide drugs, which occupy an intermediate molecular space between small molecules and proteins, constitute a therapeutic modality that offers high target specificity and low toxicity compared to conventional small-molecule and protein-based drugs. However, their clinical application is often limited by poor structural stability under physiological conditions and low cellular permeability. Moreover, their biochemical space and functional diversity are restricted to what can be achieved through combinations of the 20 canonical amino acids. To address these limitations, recent strategies, such as those employed in GLP-1–derived therapeutics, have incorporated non-canonical amino acids (ncAAs) to improve in vivo half-life. Our laboratory has developed a robust cell-free protein synthesis (CFPS) platform that enables the efficient and site-specific incorporation of ncAAs into peptides without the constraints of cellular barriers. Based on this platform, we aim to develop peptide drugs with enhanced functional properties by incorporating chemically synthesized ncAAs into biologically produced peptides, surpassing the performance of currently available drugs. Furthermore, beyond the synthesis of novel peptide scaffolds, we leverage in vitro display techniques to identify peptide binds with high affinity and specificity toward target proteins. Compared with conventional in vivo display methods, in vitro display techniques offer greater chemical diversity and substantially larger library sizes, as they are not constrained by cellular transformation efficiency or biological compatibility. As a result, they enable the iterative selection and enrichment of high-affinity peptide candidates from libraries containing 10¹²–10¹⁴ variants. For example, the RaPID platform enables the discovery of macrocyclic peptides bearing ncAAs. Collectively, this approach provides a powerful route for the identification of peptide therapeutics with enhanced binding, stability, and therapeutic potential.