Addition of nanoparticles to a cell-free transcription/translation system significantly enhanced the efficiency of the system.

Novel phosphotriesterase enzyme (PTE) monomers linked via an amino acid polypeptide are provided, expressed as a singular polypeptide with both subunits of the dimer attached from about 10 to 35 amino acids in length. This novel PTE enzyme has greater stability and/or enhanced activity in comparison to native forms of the enzyme. The novel PTE enzyme act as improved prophylactic medical counter measures against chemical nerve agents, as well as for use as decontaminants, bioscavengers for disposition in animal feedstocks, and components in assay kits.

Disclosed are mutated phosphotriesterase enzymes with improved stability and activity, as well as their use in particular for degrading organophosphorus compounds.

A genetically modified polypeptide is disclosed which comprises an amino acid sequence of phosphotriesterase (PTE) having at least twice the catalytic efficiency for a V-type nerve agent as a polypeptide which consists of the sequence as set forth in SEQ ID NO: 1, when assayed under identical conditions.

A method for designing and selecting a protein having a stabilized structure compared to a corresponding wild type protein, and proteins having at least six amino acid substitutions with respect to a corresponding wild type protein, designed for improved thermal stability, improved specific activity and/or improved expression levels, are provided herein.

Compositions and methods relating to paraoxonase fusion polypeptides are disclosed. In some aspects, the fusions are bispecific molecules that include a first biologically active polypeptide linked amino-terminal to a biologically active paraoxonase (e.g., human PON1 or a variant thereof), wherein the first biologically active polypeptide is a DNase, an RNase, a SOD1, a CTLA-4 extracellular domain, a CD40 extracellular domain, or a polypeptide that specifically binds and neutralizes an inflammatory cytokine. Bispecific fusions may further include a second biologically active polypeptide (e.g., a dimerizing domain or a domain that specifically binds to the neonatal Fc receptor (FcRn)) linked carboxyl-terminal to the first biologically active polypeptide and amino-terminal to the paraoxonase. In other aspects, a fusion polypeptide includes a biologically active paraoxonase linked carboxyl-terminal or amino-terminal to a dimerizing or FcRn-binding domain. In certain variations, a dimerizing or FcRn-binding domain is an immunoglobulin Fc region. Also disclosed are dimeric proteins comprising first and second paraoxonase fusion polypeptides as disclosed herein. The fusion polypeptides and dimeric proteins are useful in methods for therapy.

Polypeptides are disclosed which comprise an amino acid sequence of phosphotriesterase (PTE) having enhanced catalytic efficiency for VX-type or RVX-type nerve agents. Uses thereof are also disclosed.

A method for designing and selecting a protein having a stabilized structure compared to a corresponding wild type protein, and proteins having at least six amino acid substitutions with respect to a corresponding wild type protein, designed for improved thermal stability, improved specific activity and/or improved expression levels, are provided herein.

Variants of phosphotriesterase have been created that exhibit enhanced hydrolysis of V-type and G-type nerve agents over wild-type phosphotriesterase. V- and G-type nerve agents have an S.sub.P and R.sub.P enantiomer. The S.sub.P enantiomers are more toxic. V-type nerve agents are among the most toxic substances known. Variants of phosphotriesterase can prefer to hydrolyze one enantiomer of VX over the other enantiomer.

Aspects of the disclosure relate to phosphotriesterase (PTE) enzymes and PTE-related (PTER) enzymes and their use in hydrolyzing OPNAs.