Expressing a Monomeric Organophosphate Hydrolase as an EK Fusion Protein

Organophosphate hydrolase (OPH) is a bacterial paraoxonase that demonstrates wide substrate affinity against a wide range of organophosphate (OP) compounds. OPH is expressed as a stable dimeric protein in prokaryotic hosts. We demonstrate, to the best of our knowledge, the first example of a stable OPH monomeric unit by expressing a fusion protein containing alternating glutamic acid and lysine sequences (EK) at the C-terminus. This method was able to disrupt formation of the dimer interface found in OPH due to the highly hydrated and nonfouling properties of EK. This OPH-EK fusion protein demonstrated a 70% increase in catalytic activity per active site and increased substrate affinity by reducing K-m by approximately 70%. In addition, stability conferred by EK was able to overcome the stability loss caused by the elimination of the dimer interface. This strategy can potentially be used to aid in expressing prokaryotic proteins in eukaryotic hosts.