Contribution

We designed plasmids for recombinant tachypleus tridentatus clotting Factor G-consisting of an alpha and beta subunit respectively-production with E.Coli BL21 expression strain. Since this protein is derived from a eukaryotic organism, we first optimized the mature peptide sequence of each subunit for more efficient expression with a prokaryotic protein expression system and uploaded the optimized sequence of alpha and beta subunits ((BBa_K5173006, BBa_K5173007)). We designed two pET-28a vectors with the optimized sequence of the alpha or beta subunit and uploaded the two parts(BBa_K5173000, BBa_K5173001) for the reference of future teams. The two subunits would therefore be separately expressed and bind during purification to form the Factor G protein. Additionally, we inserted both optimized sequences into the two multiple cloning sites of a pET-Duet1 vector for their co-expression, which would facilitate Factor G formation, and the part (BBa_K5173002) was subsequently uploaded.

We optimized the two sequences for expression in Sf9 insect cells and uploaded the parts(BBa_K5173008, BBa_K5173009). We then designed Pfastbac1 vectors with each of the optimized sequences and uploaded the two parts(BBa_K5173004, BBa_K5173005) for the reference of future teams. Through Bac-to-Bac expression system, the two subunits would also be separately expressed and bind during purification to form the Factor G protein for future experiments.