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Efforts towards crystallization of Taq DNA polymerase mutants (967.4)

Taq DNA polymerase is an essential enzyme in biotechnological applications like the polymerase chain reaction (PCR) and Sanger sequencing. Within the last two decades, several groups have identified mutant Taq with the unnatural ability of incorporating 2’‐modified DNA, which holds implications in its use for future applications, but the origins of this activity are currently unclear. The immediate goal of this project is to develop a generalizable method for cloning, expressing and purifying Taq DNA polymerases to move forward with their crystallization and x‐ray crystal structure determination. A cloning procedure to remove undesired linker sequences present for the wt and mutant Taq plasmids has been successfully developed, and expression of these plasmids in a BL21 cell line of E. coli has been performed. The purification of enzymes by polyethyleneimine (PEI) precipitation and fast pressure liquid chromatography (FPLC) is currently being optimized. Finalizing these procedures will provide the groundwork for using x‐ray crystallography to determine and compare the structures of wild type (wt) and mutant Taq while incorporating modified and unmodified nucleotides, which in turn will aid in future protein‐engineering efforts.