Subject(s)
Folate Receptors, GPI-Anchored/metabolism , T-Lymphocytes, Cytotoxic/immunology , Animals , CD3 Complex/immunology , Cell Line, Tumor , Coculture Techniques , Folic Acid/chemistry , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin Fc Fragments/immunology , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Immunotherapy , Leukocytes, Mononuclear/cytology , Male , Mice , Mice, Inbred NOD , Mice, SCID , Neoplasms/metabolism , Neoplasms/pathology , Neoplasms/therapy , Rats , Rats, Sprague-Dawley , T-Lymphocytes, Cytotoxic/cytologyABSTRACT
A procedure for the analysis of short-chain intracellular coenzyme A (CoA) esters and adenine nucleotide pools in microbial cells is described. The simultaneous isolation of bacterial cells from media, quenching of their metabolism, and extraction of metabolites was accomplished by centrifugation of cells through a layer of silicone oil into a denser solution of trichloroacetic acid. The acid was neutralized by extraction into Freon containing tri-n-octylamine to provide a salt-free solution of cell metabolites. After high-performance liquid chromatography separation, CoA, CoA esters, and adenine-containing nucleotides were derivatized by postcolumn reaction with bromoacetaldehyde to form the fluorescent 1,N6-ethenoadenine adducts which were analyzed by a fluorescence detector at picomolar levels.
Subject(s)
Coenzyme A/analysis , Chromatography, High Pressure Liquid , Coenzyme A/metabolism , Escherichia coli/metabolism , Esters , Time FactorsABSTRACT
A surface anchor system derived from the ice-nucleation protein (INP) from Pseudomonas syringe was used to localize organophosphorus hydrolase (OPH) onto the surface of Pseudomonas putida KT2440. Cells harboring the shuttle vector pPNCO33 coding for the INP-OPH fusion were capable of targeting OPH onto the cell surface as demonstrated by whole cell ELISA. The whole cell activity of P. putida KT2440 was shown to be 10 times higher than those of previous efforts expressing the same fusion protein in Escherichia coli. The capability of expressing enzymes on the surface of a robust and environmentally benign P. putida KT2440 should open up new avenues for a wide range of applications such as in situ bioremediation.
Subject(s)
Aryldialkylphosphatase/biosynthesis , Bacterial Outer Membrane Proteins/metabolism , Cell Membrane/enzymology , Protein Engineering/methods , Pseudomonas putida/enzymology , Aryldialkylphosphatase/chemistry , Aryldialkylphosphatase/genetics , Bacterial Outer Membrane Proteins/genetics , Biodegradation, Environmental , Enzyme Activation , Gene Expression Regulation, Bacterial/physiology , Gene Expression Regulation, Enzymologic/physiology , Membrane Proteins/genetics , Membrane Proteins/metabolism , Pesticides/pharmacokinetics , Protein Binding , Pseudomonas putida/genetics , Pseudomonas putida/growth & development , Recombinant Fusion Proteins/metabolism , Soil Pollutants/pharmacokineticsABSTRACT
A bifunctional fusion protein consisting of organophosphorus hydrolase (OPH) and elastin-like polypeptide (ELP) was synthesized for the detoxification of organophosphorus compounds. ELPs undergo a reversible phase transition upon an increase in temperature, forming hydrophobic aggregates. This thermally triggered property of phase transition allows for a simple and rapid means of purifying the fusion protein. Over 1,300-fold purification was achieved after only 2 cycles of inverse phase transition. The purified fusion protein showed identical kinetic properties as the native OPH with only a modest 10% increase in K(m) and a 5% decrease of K(cat). The ability of the ELP domain to form collapsed aggregates also improved long-term stability of the fusion enzyme. Aggregated ELP-OPH retained nearly 100% activity over a span of three weeks. In addition to facilitating purification and stability, the ELP moiety served as a hydrophobic tag for one-step immobilization of the fusion protein onto hydrophobic surfaces. The ELP-OPH was capable of rapidly degrading paraoxon while immobilized. The protein also retained ELP functionality of reversible phase transition thereby allowing for the regeneration of the treated surface. This technology offers a swift and convenient means for purification, immobilization, and regeneration of OPH onto a variety of hydrophobic surfaces by simple environmental triggers.
