PT - JOURNAL ARTICLE AU - Homma, Toshimasa AU - Yamamoto, Rie AU - Ang, Lily Zuin Ping AU - Fehaid, Alaa AU - Ebara, Mitsuhiro TI - A Novel Gene Synthesis Platform for Designing Functional Protein Polymers AID - 10.1101/2024.09.01.610679 DP - 2024 Jan 01 TA - bioRxiv PG - 2024.09.01.610679 4099 - http://biorxiv.org/content/early/2024/09/03/2024.09.01.610679.short 4100 - http://biorxiv.org/content/early/2024/09/03/2024.09.01.610679.full AB - Recombinant protein polymers with repeat sequences of specific amino acids can be regarded as sustainable functional materials that can be designed using genetic engineering. However, synthesizing genes encoding these proteins is significantly time-consuming and labor-intensive owing to the difficulty of using common gene synthesis tools, such as restriction enzymes and PCR primers. To overcome these obstacles, we propose a novel method: seamless cloning of rolling-circle amplicons (SCRCA). This method involves one-pot preparation of repetitive-sequence genes with overlapping ends for cloning, facilitating the easy construction of the desired recombinants. Using SCRCA, we synthesized 10 genes encoding hydrophilic resilin-like and hydrophobic elastin-like repeat units that induce liquid–liquid phase separation. SCRCA shows higher transformation efficiency and better workability than conventional methods, and the time and budget required for SCRCA are comparable to those required for non-repetitive-sequence gene synthesis. Additionally, SCRCA allows the construction of a repeat unit library at a low cost. The library shows considerably higher diversity compared with that of the state-of-the-art method. By combining this library construction with the directed evolution concept, we can rapidly develop an elastin-like protein polymer with a desired function. SCRCA can greatly accelerate research on protein polymers.Competing Interest StatementThe National Institute of Technology has filed a patent application on the repetitive-sequence gene library construction (PCT/JP2023/014202). The National Institute of Technology and NIMS have filed another patent application on the gene synthesis method. All other authors declare no competing interests.