Spider silk spidroins, nature's advanced polymers, have long hampered efficient in vitro production due to their considerable size, repetitive sequences, and aggregation-prone nature. This study harnesses the power of a cell-free protein synthesis (CFPS) system, presenting the first successful in vitro production and detailed characterization of recombinant spider silk major ampullate spidroins (MaSps) utilizing a reformulated and optimizedEscherichia coli based CFPS system. Through systematic optimization, including cell strain engineering via knockout generation, energy sources, crowding agents, and amino acid supplementation, we effectively addressed the specific challenges associated with recombinant spidroin biosynthesis, resulting in high yields of 0.61 mg/mL for MaSp1 (69 kDa) and 0.52 mg/mL for MaSp2 (73 kDa). The synthesized spidroins self-assembled into micelles, facilitating efficient purification compared to in vivo methods, and were further processed into prototype silk fiber products. The functional characterization demonstrated that the purified spidroins maintain essential natural properties, such as phase separation and fiber formation triggered by pH and ions. This tailored CFPS platform also facilitates versatile cosynthesis and serves as an accessible platform for studying the supramolecular coassembly and dynamic interactions among spidroins. This CFPS platform offers a viable alternative to conventional in vivo methods, facilitating innovative approaches for silk protein engineering and biomaterial development in a high-throughput, efficient manner.
Keywords: cell-free protein synthesis; fiber; phase separation; self-assembly; spider silk; spidroin.