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51.
Comparison of the crystal structures of genetically engineered human manganese superoxide dismutase and manganese superoxide dismutase from Thermus thermophilus: differences in dimer-dimer interaction.
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U. G. Wagner K. A. Pattridge M. L. Ludwig W. C. Stallings M. M. Werber C. Oefner F. Frolow J. L. Sussman 《Protein science : a publication of the Protein Society》1993,2(5):814-825
The three-dimensional X-ray structure of a recombinant human mitochondrial manganese superoxide dismutase (MnSOD) (chain length 198 residues) was determined by the method of molecular replacement using the related structure of MnSOD from Thermus thermophilus as a search model. This tetrameric human MnSOD crystallizes in space group P2(1)2(1)2 with a dimer in the asymmetric unit (Wagner, U.G., Werber, M.M., Beck, Y., Hartman, J.R., Frolow, F., & Sussman, J.L., 1989, J. Mol. Biol. 206, 787-788). Refinement of the protein structure (3,148 atoms with Mn and no solvents), with restraints maintaining noncrystallographic symmetry, converged at an R-factor of 0.207 using all data from 8.0 to 3.2 A resolution and group thermal parameters. The monomer-monomer interactions typical of bacterial Fe- and Mn-containing SODs are retained in the human enzyme, but the dimer-dimer interactions that form the tetramer are very different from those found in the structure of MnSOD from T. thermophilus. In human MnSOD one of the dimers is rotated by 84 degrees relative to its equivalent in the thermophile enzyme. As a result the monomers are arranged in an approximately tetrahedral array, the dimer-dimer packing is more intimate than observed in the bacterial MnSOD from T. thermophilus, and the dimers interdigitate. The metal-ligand interactions, determined by refinement and verified by computation of omit maps, are identical to those observed in T. thermophilus MnSOD. 相似文献
52.
Julia Schollmeyer Saskia Waldburger Kendra Njo Heba Yehia Anke Kurreck Peter Neubauer Sebastian L. Riedel 《Biotechnology and bioengineering》2023,120(11):3322-3334
Nucleoside phosphorylases are important biocatalysts for the chemo-enzymatic synthesis of nucleosides and their analogs which are, among others, used for the treatment of viral infections or cancer. S-methyl-5′-thioadenosine phosphorylases (MTAP) are a group of nucleoside phosphorylases and the thermostable MTAP of Aeropyrum pernix (ApMTAP) was described to accept a wide range of modified nucleosides as substrates. Therefore, it is an interesting biocatalyst for the synthesis of nucleoside analogs for industrial and therapeutic applications. To date, thermostable nucleoside phosphorylases were produced in shake flask cultivations using complex media. The drawback of this approach is low volumetric protein yields which hamper the wide-spread application of the thermostable nucleoside phosphorylases in large scale. High cell density (HCD) cultivations allow the production of recombinant proteins with high volumetric yields, as final optical densities >100 can be achieved. Therefore, in this study, we developed a suitable protocol for HCD cultivations of ApMTAP. Initially, optimum expression conditions were determined in 24-well plates using a fed-batch medium. Subsequently, HCD cultivations were performed using E. coli BL21-Gold cells, by employing a glucose-limited fed-batch strategy. Comparing different growth rates in stirred-tank bioreactors, cultivations revealed that growth at maximum growth rates until induction resulted in the highest yields of ApMTAP. On a 500-mL scale, final cell dry weights of 87.1–90.1 g L−1 were observed together with an overproduction of ApMTAP in a 1.9%–3.8% ratio of total protein. Compared to initially applied shake flask cultivations with terrific broth (TB) medium the volumetric yield increased by a factor of 136. After the purification of ApMTAP via heat treatment and affinity chromatography, a purity of more than 90% was determined. Activity testing revealed specific activities in the range of 0.21 ± 0.11 (low growth rate) to 3.99 ± 1.02 U mg−1 (growth at maximum growth rate). Hence, growth at maximum growth rate led to both an increased expression of the target protein and an increased specific enzyme activity. This study paves the way towards the application of thermostable nucleoside phosphorylases in industrial applications due to an improved heterologous expression in Escherichia coli. 相似文献
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Recombinant isopenicillin N synthase fromStreptomyces clavuligerus was produced in the form of inactive inclusion bodies inEscherichia coli. These inclusion bodies were solubilized by treatment with 5 M urea under reducing conditions. Optimization of refolding conditions to recover active isopenicillin N synthase indicated that a dialysis procedure carried out at a protein concentration of about 1.0 mg ml–1 gave maximal recovery of active isopenicillin N synthase. Solubilized isopenicillin N synthase of more than 95% purity was obtained by passing this material through a DEAE-Trisacryl ion exchange column. Expression studies conducted at different temperatures indicated that isopenicillin N synthase was produced predominantly in a soluble, active form when expression was conducted at 20°C, and accounted for about 20% of the total soluble protein. This high-level production facilitated the purification of soluble isopenicillin N synthase to near homogeneity in four steps. Characterization of the purified soluble and solubilized isopenicillin N synthase revealed that they are very similar. 相似文献
56.
TheAnticarsia gemmatalisnuclear polyhedrosis virus (AgNPV) was released in two soybean plots in September, 1991; the soil in the plots was then sampled periodically through July, 1992, to determine the effects of normal agricultural soil manipulations and precipitation on vertical distribution of the polyhedral occlusion bodies (POBs). The amount of AgNPV increased at all depths down to 37.5–50 cm as long as there was virus-contaminated plant matter, even dead soybean refuse, above the soil surface. Agricultural operations (disking, harrowing, mowing, planting, cultivating) had no effect on the amount or vertical distribution of AgNPV in soil. After the crop refuse was disked into the soil in November, the amount of POBs began decreasing at all depths; these decreases continued over winter and at times appeared to be associated with precipitation. The POBs were no longer detected below 37.5 cm by April, 1992, or below 25 cm by July, 1992. However, in July there were still 274 POBs/g in the top 2.5 cm of soil. Thus, agricultural operations should not hinder contamination of soybean leaves by AgNPV from its soil reservoir. 相似文献
57.
Maurizi G Bossù P Boraschi D Ulisse E Tagliabue A Ruggiero P 《Biotechnology and bioengineering》1995,48(3):197-200
A recombinant strain of Bacillus subtilis engineered for endocellular expression of human interleukin-1 receptor antagonist (IL-Ira) was subjected to sporulation. The recombinant protein was recovered from the sporulation supernatant in quantities, purity, and activity comparable with those obtained from a traditional cell lysate. Thus, exploitation of this natural mechanism of autolysis could overcome problems of intact protein recovery related to the cell disruption step. (c) 1995 John Wiley & Sons, Inc. 相似文献
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59.
During the production by mammalian cells of recombinant factor VIII from which the B domain was deleted (rFVIII), proteolytic
cleavages in the C-terminal part of the heavy chain were observed (Kjalke et al., 1995). By radioactive pulse labelling it
was investigated whether the cleavages took place inside the cells during protein synthesis or after release in the medium.
The rFVIII-producing CHO (Chinese hamster ovary) cells were cultured in the presence of 35S-methionine and then the cell lysate and the conditioned media were immunoprecipitated and analyzed by electrophoresis. By
pulse labelling and chasing for various time periods, it was shown that the cleavages only took place after secretion of the
protein from the cells. Adding cell lysate to uncleaved rFVIII caused cleavage of the heavy chain, as seen by loss of binding
to a monoclonal antibody specific for intact rFVIII, indicating that the cleavage was performed by proteinase(s) released
from the lysed cells. By incubating intact rFVIII with the multicatalytic proteinase (proteasome) present in cytoplasm and
nucleus of eukaryotic cells, loss of binding to the monoclonal antibody was observed. This indicates that the multicatalytic
proteinase, released from lysed rFVIII producing cells, could be responsible for the cleavage of rFVIII. Among several protease
inhibitors tested, only bacitracin was found to diminish the extent of cleavage. Phosphatidylserine also protected rFVIII
against cleavage, probably by binding to rFVIII.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
60.
The maltose binding protein (MBP or MalE) of Escherichia coli is the periplasmic component of the transport system for malto-oligosaccharides. It is used widely as a carrier protein for the production of recombinant fusion proteins. The melting of recombinant MBP was studied by differential scanning and titration calorimetry and fluorescence spectroscopy under different solvent conditions. MBP exhibits a single peak of heat absorption with a delta(Hcal)/delta(HvH) ratio in the range of 1.3-1.5, suggesting that the protein comprises two strongly interacting thermodynamic domains. Binding of maltose resulted in elevation of the Tm by 8-15 degrees C, depending of pH. The presence of ligand at neutral pH, in addition to shifting the melting process to higher temperature, caused it to become more cooperative. The delta(Hcal)/delta(HvH) ratio decreased to unity, indicating that the two domains melt together in a single two-state transition. This ligand-induced merging of the two domains appears to occur only at neutral pH, because at low pH maltose simply stabilized MBP and did not cause a decrease of the delta(Hcal)/delta(HvH) ratio. Binding of maltose to MBP is characterized by very low enthalpy changes, approximately -1 kcal/mol. The melting of MBP is accompanied by an exceptionally large change in heat capacity. 0.16 cal/K-g, which is consistent with the high amount of nonpolar surface--0.72 A2/g--that becomes accessible to solvent in the unfolded state. The high value of delta Cp determines a very steep delta G versus T profile for this protein and predicts that cold denaturation should occur above freezing temperatures. Evidence for this was provided by changes in fluorescence intensity upon cooling the protein. A sigmoidal cooperative transition with a midpoint near 5 degrees C was observed when MBP was cooled at low pH. Analysis of the melting of several fusion proteins containing MBP illustrated the feasibility of assessing the folding integrity of recombinant products prior to separating them from the MBP carrier protein. 相似文献