(715d) Recombinant Spider Silk Protein Production in Escherichia Coli Cell-Free Protein Synthesis

Spider dragline silk is a tough natural fiber that promises numerous industrial and biomedical applications. Producing native-sized recombinant spider silk (>250 kDa) has been limited because of difficulties in translation due to unbalanced tRNA pools and the extremely long template. Recently, these challenges were addressed for the first time in vivo by engineering an Escherichia coli strain with an elevated glycyl-tRNA pool. Here, we sought to express native-sized spider silk using cell-free protein synthesis (CFPS). In CFPS, components can be tuned to unnatural levels, new components (natural and unnatural) can be added or synthesized and can be maintained at precise ratios, and the chemical environment can be controlled, actively monitored, and rapidly sampled. We targeted the synthesis of two spider silk-like proteins, the major ampullate spidroins 1 and 2 (MaSp1 and MaSp2). We constructed plasmid templates of different repeat sizes of spider silk gene ranging 4 to 128 repeats via multiple rounds of recursive cloning and tested in CFPS using E. coli cell extract. Synthesis of spider silk protein was detected up to 64 repeats (170 kDa). To our knowledge, this is largest polypeptide expressed using bacterial CFPS. The addition of excess glycyl-tRNA and glycine improved spider silk protein production. Beyond improvements afforded by tuning the components of translation, I will also discuss our efforts to incorporate unnatural amino acids into native-sized spider silk proteins by amber suppression in extracts from genomically recoded organisms (GROs) for introducing new functional properties. Looking forward, we anticipate that protein-like sequence defined polymers hold promise as a new class of high performance, lightweight, and innovative materials. Moreover, we expect that CFPS will provide a highly tunable approach for the production of genetically encoded materials that are difficult to produce in vivo.