Three native E. coli proteins-NusA, GrpE, and bacterioferritin (BFR)-were studied in fusion proteins expressed in E. coli for their ability to confer solubility on a target insoluble protein at the C-terminus of the fusion protein. These three proteins were chosen based on their favorable cytoplasmic solubility characteristics as predicted by a statistical solubility model for recombinant proteins in E. coli. Modeling predicted the probability of soluble fusion protein expression for the target insoluble protein human interleukin-3 (hIL-3) in the following order: NusA (most soluble), GrpE, BFR, and thioredoxin (least soluble). Expression experiments at 37 degrees C showed that the NusA/hIL-3 fusion protein was expressed almost completely in the soluble fraction, while GrpE/hIL-3 and BFR/hIL-3 exhibited partial solubility at 37 degrees C. Thioredoxin/hIL-3 was expressed almost completely in the insoluble fraction. Fusion proteins consisting of NusA and either bovine growth hormone or human interferon-gamma were also expressed in E. coli at 37 degrees C and again showed that the fusion protein was almost completely soluble. Starting with the NusA/hIL-3 fusion protein with an N-terminal histidine tag, purified hIL-3 with full biological activity was obtained using immobilized metal affinity chromatography, factor Xa protease cleavage, and anion exchange chromatography.
Catalytic alkane functionalization by the Fe(TPA)/tBuOOH system (with [Fe(TPA)Cl2]+ (1), [Fe(TPA)Br2]+ (2), and [Fe2O(TPA)2(H2O)2]4+ (3) as catalysts; TPA = tris(2-pyridylmethyl)amine) has been investigated in further detail to clarify whether the reaction mechanism involves a metal-based oxidation or a radical chain autoxidation. These two mechanisms can be distinguished by the nature of the products formed, their dependence on O2 (determined from argon purge and 18O2 labeling experiments), and the kinetic isotope effects associated with the products. The metal-based oxidation mechanism is analogous to heme-catalyzed hydroxylations and would be expected to produce mostly alcohol with a large kinetic isotope effect. The radical chain autoxidation mechanism entails the trapping of substrate alkyl radicals by O2 to afford alkylperoxy radicals that decompose to alcohol and ketone products in a ratio 1:1 or smaller via Russell termination steps. Consistent with the latter mechanism, alcohol and ketone products were observed in a ratio of 1:1 or less, when catalysts 1, 2, or 3 were reacted with alkane and 150 equiv of tBuOOH; these product yields were diminished by argon purging, demonstrating the participation of O2 in the reaction. However, when the 3-catalyzed oxidation was carried out in the presence of a limited (<20 equiv) amount of tBuOOH or CmOOH, the sole product observed was alcohol; k H/k D values of 10 were observed, consistent with a metal-based oxidation. To reconcile these apparently conflicting results, a mechanistic scheme is proposed involving the formation of an alkylperoxyiron(III) intermediate which can oxidize either the substrate (metal-based oxidation) or excess ROOH (to generate alkylperoxy radicals that initiate a radical chain autoxidation process), the relative importance of the two mechanisms being determined by the concentration of ROOH.
T2 relaxation makes an important contribution to tissue contrast in magnetic resonance (MR) imaging. Many tissues are known to exhibit multicomponent T2 relaxation that suggests some compartmental segregation of mobile protons on a T2 timescale. Magnetization transfer (MT) is another relaxation mechanism that can be used to produce tissue contrast in MR imaging. The MT process depends strongly on water-macromolecular interactions. To investigate the relationship between multicomponent T2 relaxation and the MT process, multiecho T2 measurements have been combined with MT measurements for freshly excised samples of cardiac muscle, striated muscle, and white matter. For muscle, short T2 components show greater MT than long T2 components, consistent with the belief that they represent distinct water environments. For white matter, quantitative MT measurements were identical for the two major T2 components, apparently because of exchange between the T2 compartments on a time-scale characteristic of the MT experiment. Implications for accurate modeling of MT in tissue and the use of MT for MR image contrast are discussed.
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