A Novel Gene Synthesis Platform for Designing Functional Protein Polymers.

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, a novel method is proposed herein: 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. SCRCA is used to synthesize 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 facilitates the construction of a repeat unit library at a low cost. The library shows considerably higher diversity than that of the current state-of-the-art method. By combining this library construction with the directed evolution concept, an elastin-like protein polymer with the desired functions can be rapidly developed. SCRCA can greatly accelerate research on protein polymers.