Recombinant protein expression and purification of codon-optimized Pfu-Sso7d v2

This protocol has been optimized for the recombinant expression of a codon-optizimed Pfu-Sso7d DNA polymerase. This is a fusion protein composed of the Pfu enzyme from Pyrococcus furiosus for DNA amplification by PCR fused to a small 7 kDa protein from Sulfobulus solfataricus that binds to double-stranded DNA without any preference for specific sequences, thus enhancing polymerization processivity without affecting the catalytic activity or thermal stability of the enzyme. The goal of this protocol was to eliminate the use of large volumes for dyalisis and potential issues with the protein crashing out of the solution due to the use of concentrators for buffer exchange of this enzyme into storage conditions. We also eliminated the use of DTT, which is often found in other similar protocols. The sequence plasmid encoding the codon-optimized Pfu-Sso7d enzyme used here can be found at https://benchling.com/s/seq-2TcUPjO2uMbDG5ufTQN4