Association of partially-folded intermediates of staphylococcal nuclease induces structure and stability.

Staphylococcal nuclease forms three different partially-folded intermediates at low pH in the presence of low to moderate concentration of anions, differing in the amount of secondary structure, globularity, stability, and compactness. Although these intermediates are monomeric at low protein concentration (< or =0.25 mg/mL), increasing concentrations of protein result in the formation of dimers and soluble oligomers, ultimately leading to larger insoluble aggregates. Unexpectedly, increasing protein concentration not only led to association, but also to increased structure of the intermediates. The secondary structure, stability, and globularity of the two less-ordered partially-folded intermediates (A1 and A2) were substantially increased upon association, suggesting that aggregation induces structure. An excellent correlation was found between degree of association and amount of structure measured by different techniques, including circular dichroism, fluorescence, Fourier transform infrared spectroscopy (FTIR), and small-angle X-ray scattering. The associated states were also substantially more stable toward urea denaturation than the monomeric forms. A mechanism is proposed, in which the observed association of monomeric intermediates involves intermolecular interactions which correspond to those found intramolecularly in normal folding to the native state

Characterization of Aryloxyalkanoate Dioxygenase-12, a Nonheme Fe(II)/α-Ketoglutarate-Dependent Dioxygenase, Expressed in Transgenic Soybean and <i>Pseudomonas fluorescens</i>

Aryloxyalkanoate dioxygenase-12 (AAD-12) was discovered from the soil bacterium <i>Delftia acidovorans</i> MC1 and is a nonheme Fe­(II)/α-ketoglutarate-dependent dioxygenase, which can impart herbicide tolerance to transgenic plants by catalyzing the degradation of certain phenoxyacetate, pyridyloxyacetate, and aryloxyphenoxypropionate herbicides. The development of commercial herbicide-tolerant crops, in particular AAD-12-containing soybean, has prompted the need for large quantities of the enzyme for safety testing. To accomplish this, the enzyme was produced in <i>Pseudomonas fluorescens</i> (<i>Pf</i>) and purified to near homogeneity. A small amount of AAD-12 was partially purified from transgenic soybean and through various analytical, biochemical, and <i>in vitro</i> activity analyses demonstrated to be equivalent to the <i>Pf</i>-generated enzyme. Furthermore, results from <i>in vitro</i> kinetic analyses using a variety of plant endogenous compounds revealed activity with <i>trans</i>-cinnamate and indole-3-acetic acid (IAA). The catalytic efficiencies (<i>k</i>_cat/<i>K</i>_m) of AAD-12 using <i>trans</i>-cinnamate (51.5 M^–1 s^–1) and IAA (8.2 M^–1 s^–1) as substrates were very poor when compared to the efficiencies of plant endogenous enzymes. The results suggest that the presence of AAD-12 in transgenic soybean would not likely have an impact on major plant metabolic pathways

An antibody produced in tobacco expressing a hybrid β-1,4-galactosyltransferase is essentially devoid of plant carbohydrate epitopes

N-glycosylation of a mAb may have a major impact on its therapeutic merits. Here, we demonstrate that expression of a hybrid enzyme (called xylGalT), consisting of the N-terminal domain of Arabidopsis thaliana xylosyltransferase and the catalytic domain of human β-1,4-galactosyltransferase I (GalT), in tobacco causes a sharp reduction of N-glycans with potentially immunogenic core-bound xylose (Xyl) and fucose (Fuc) residues as shown by Western blot and MALDI-TOF MS analysis. A radioallergosorbent test inhibition assay with proteins purified from leaves of WT and these transgenic tobacco plants using sera from allergic patients suggests a significant reduction of potential immunogenicity of xylGalT proteins. A mAb purified from leaves of plants expressing xylGalT displayed an N-glycan profile that featured high levels of galactose, undetectable xylose, and a trace of fucose. Hence, a transgenic plant expressing the hybrid GalT might yield more effective and safer monoclonals for therapeutic purposes than WT plants and even transgenic plants expressing the unchanged GalT