Organophosphate Hydrolase (OPH) Designed as a Functional Monomer
Abstract
Organophosphate hydrolase (OPH) mutants haveshown potential use as a medical countermeasure against a range of differentorganophosphorus compounds (OPs). OPH is typicallyexpressed in bacteria as a homodimer. Two separate monomers (~37kDa each) self‐assemble throughnon‐covalent bonding at the enzyme face close to the putative active site, andenzyme activity is dependent upon dimer formation. OPH homodimers do notsecrete expediently from mammalian cells and are often misfolded. This causespotential problems when trying to express the protein from a heterologous plasmidor viral delivery system. To enhance secretion of OPH from mammalian cells, wesought to increase protein solubility without catastrophic detriment toactivity and without addition of fusion proteins. To this end, we designed OPH tobe expressed as a functional monomer by joining two OPH subunits with a poly‐glycinelinker and expressing it as a single polypeptide in bacteria to evaluateactivity. We created the single polypeptide OPH with a tether 10 or 35 aminoacids in length between the two halves, and named them T10 and T35, respectively. Western blot analysis and paraoxon hydrolysis assays revealedthat T10 was being produced and there was a 71% loss in specific activity comparedto an untethered homodimer positive control. This was a surprise as we expectedT10 to have no enzymatic activity since the tether was too short to allowproper dimer formation. The T35 functional monomer (~76 kDa) was produced showing 29% loss of specific activity. Both T10 and T35 OPH showed high molecular weight aggregate formation greater than 250 kDa in dynamic light scattering and native polyacrylamide gels. Construction of a functional OPH monomer with activity against OPs is instrumental in designing secretable OPHs for adeno‐associatedviral expression. It may also prove useful in the development of chimeric OPHs for maintaining control over the composition of each OPH half, allowing blended activities against a combination of organophosphorus substrates. This is the first attempt known for producing OPH as a functional monomer.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Apr 01, 2018
- Source ID
- 10.1096/fasebj.2018.32.1_supplement.lb66
Entities
People
- Cetara A Baker
- Jaffet Santiago Garcia
- Richard Sweeney
- Stephen Kirby
Organizations
- Oak Ridge Institute for Science and Education
- United States Army Medical Research Institute of Chemical Defense
- United States Department of Defense
- United States Department of Energy