Anti-His tag antibody

Objectives To survey a library of over-expressed nitroreductases to identify those most active with 2,4- and 2,6-dinitrotoluene substrates, as promising candidates for phytoremediation of soils and groundwater contaminated with poly-nitro toluene pollutants. Results To indirectly monitor dinitrotoluene reduction we implemented a nitroblue tetrazolium dye screen to compare relative rates of NADPH consumption for 58 nitroreductase candidates, over-expressed in a nitroreductase-deleted strain of Escherichia coli. Although the screen only provides activity data at a single substrate concentration, by altering the substrate concentration and duration of incubation we showed we could first distinguish between more-active and less-active enzymes and then discriminate between the relative rates of reduction exhibited by the most active nitroreductases in the collection. We observed that members of the NfsA and NfsB nitroreductase families were the most active with 2,4-dinitrotoluene, but that only members of the NfsB family reduced 2,6-dinitrotoluene effectively. Two NfsB family members, YfkO from Bacillus subtilis and NfsB from Vibrio vulnificus, appeared especially effective with these substrates. Purification of both enzymes as His(6)-tagged recombinant proteins enabled in vitro determination of Michaelis-Menten kinetic parameters with each dinitrotoluene substrate. Conclusions Vibrio vulnificus NfsB is a particularly promising candidate for bioremediation applications, being ca. fivefold more catalytically efficient with 2,4-dinitrotoluene and over 26-fold more active with 2,6-dinitrotoluene than the benchmark E. coli nitroreductases NfsA and NfsB.

In this study, core-shell Fe3O4@Au nanoparticles with good chemical stability and tunable particle size were synthesized via Au-S bonding, which overcomes the challenges encountered for the reported preparation of Fe3O4@Au. The surface of nanoparticles was investigated by TEM, XRD, XPS, FT-IR, VSM and DLS which characterized the size, morphology, chemical lattice, elemental analysis, functional groups, magnetic strength, and size distribution. The results showed that Fe3O4@Au/NTA-Ni2+ magnetic nanocomposites were spherical with an average diameter of 469.5nm, and superparamagnetic with saturation magnetization of 7 emu/g. The magnetic nanocomposites were directly utilized for one-step purification of His-tagged proteins from Description Escherichia coli lysate. The as-separated proteins were qualitatively validated by gel analysis and quantitatively measured by UV-visible spectroscopy. The binding capacity of Fe3O4@Au/NTA-Ni2+ towards His-tagged proteins was 48.3 mg/g. The target proteins were separated with high purity and separation efficiency up to 96.6%. Their specificity and enrichment ability were maintained during six cycles of adsorption-elution operations. The advantages of this novel nanoparticle can open a window for the multifunction of nanomaterials and their biological application.