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The Escherichia coli protein HU is a non-sequence-specific DNA-binding protein that interacts with DNA primarily through electrostatic interactions. In addition to nonspecific binding to linear DNA, HU has been shown to bind with nanomolar affinity to discontinuous DNA substrates, such as repair and recombination intermediates. This work specifically examines the HU-four-way junction (4WJ) interaction using fluorescence spectroscopic methods. The conformation of the junction in the presence of different counterions was investigated by Fo?rster resonance energy transfer (FRET) measurements, which revealed an ion-type conformational dependence, where Na(+) yields the most stacked conformation followed by K(+) and Mg(2+). HU binding induces a greater degree of stacking in the Na(+)-stabilized and Mg(2+)-stabilized junctions but not the K(+)-stabilized junction, which is attributed to differences in the size of the ionic radii and potential differences in ion binding sites. Interestingly, junction conformation modulates binding affinity, where HU exhibits the lowest affinity for the Mg(2+)-stabilized form (24 μM(-1)), which is the least stacked conformation. Protein binding to a mixed population of open and stacked forms of the junction leads to nearly complete formation of a protein-stabilized stacked-X junction. These results strongly support a model in which HU binds to and stabilizes the stacked-X conformation. 相似文献
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Ignea Codruta Trikka Fotini A Kourtzelis Ioannis Argiriou Anagnostis Kanellis Angelos K Kampranis Sotirios C Makris Antonios M 《Microbial cell factories》2012,11(1):1-16
Background
Production of correctly disulfide bonded proteins to high yields remains a challenge. Recombinant protein expression in Escherichia coli is the popular choice, especially within the research community. While there is an ever growing demand for new expression strains, few strains are dedicated to post-translational modifications, such as disulfide bond formation. Thus, new protein expression strains must be engineered and the parameters involved in producing disulfide bonded proteins must be understood.Results
We have engineered a new E. coli protein expression strain named SHuffle, dedicated to producing correctly disulfide bonded active proteins to high yields within its cytoplasm. This strain is based on the trxB gor suppressor strain SMG96 where its cytoplasmic reductive pathways have been diminished, allowing for the formation of disulfide bonds in the cytoplasm. We have further engineered a major improvement by integrating into its chromosome a signal sequenceless disulfide bond isomerase, DsbC. We probed the redox state of DsbC in the oxidizing cytoplasm and evaluated its role in assisting the formation of correctly folded multi-disulfide bonded proteins. We optimized protein expression conditions, varying temperature, induction conditions, strain background and the co-expression of various helper proteins. We found that temperature has the biggest impact on improving yields and that the E. coli B strain background of this strain was superior to the K12 version. We also discovered that auto-expression of substrate target proteins using this strain resulted in higher yields of active pure protein. Finally, we found that co-expression of mutant thioredoxins and PDI homologs improved yields of various substrate proteins.Conclusions
This work is the first extensive characterization of the trxB gor suppressor strain. The results presented should help researchers design the appropriate protein expression conditions using SHuffle strains. 相似文献4.
Brandusa Elena Lixandru Ani Ioana Cotar Monica Straut Codruta Romanita Usein Dana Cristea Simona Ciontea Dorina Tatu-Chitoiu Irina Codita Alexandru Rafila Maria Nica Mariana Buzea Anda Baicus Mihaela Camelia Ghita Irina Nistor Cristina Tuchilu? Marina Indreas Felicia Antohe Corinna Glasner Hajo Grundmann Aftab Jasir Maria Damian 《PloS one》2015,10(11)
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Codruta?Ignea Ivana?Cvetkovic Sofia?Loupassaki Panagiotis?Kefalas Christopher?B?Johnson Sotirios?C?Kampranis Antonios?M?MakrisEmail author 《Microbial cell factories》2011,10(1):4
Background
Terpenoids constitute a large family of natural products, attracting commercial interest for a variety of uses as flavours, fragrances, drugs and alternative fuels. Saccharomyces cerevisiae offers a versatile cell factory, as the precursors of terpenoid biosynthesis are naturally synthesized by the sterol biosynthetic pathway. 相似文献6.
Removing heavy metals from synthetic effluents using “kamikaze” Saccharomyces cerevisiae cells 总被引:1,自引:0,他引:1
Lavinia Ruta Codruta Paraschivescu Mihaela Matache Sorin Avramescu Ileana Cornelia Farcasanu 《Applied microbiology and biotechnology》2010,85(3):763-771
One key step of the bioremediation processes designed to clean up heavy metal contaminated environments is growing resistant
cells that accumulate the heavy metals to ensure better removal through a combination of biosorption and continuous metabolic
uptake after physical adsorption. Saccharomyces cerevisiae cells can easily act as cation biosorbents, but isolation of mutants that are both hyperaccumulating and tolerant to heavy
metals proved extremely difficult. Instead, mutants that are hypersensitive to heavy metals due to increased and continuous
uptake from the environment were considered, aiming to use such mutants to reduce the heavy metal content of contaminated
waters. In this study, the heavy metal hypersensitive yeast strain pmr1∆ was investigated for the ability to remove Mn2+, Cu2+, Co2+, or Cd2+ from synthetic effluents. Due to increased metal accumulation, the mutant strain was more efficient than the wild-type in
removing Mn2+, Cu2+, or Co2+ from synthetic effluents containing 1–2 mM cations, with a selectivity $ {\text{Mn}}^{{{\text{2}} + }} > {\text{Co}}^{{{\text{2}} + }} ~ > {\text{Cu}}^{{{\text{2}} + }} $ {\text{Mn}}^{{{\text{2}} + }} > {\text{Co}}^{{{\text{2}} + }} ~ > {\text{Cu}}^{{{\text{2}} + }} and also in removing Mn2+ and Cd2+ from synthetic effluents containing 20–50 μM cations, with a selectivity Mn2+ > Cd2+. 相似文献
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Dumitru I Ene CD Ofiteru AM Paraschivescu C Madalan AM Baciu I Farcasanu IC 《Journal of biological inorganic chemistry》2012,17(6):961-974
Superoxide dismutases (SODs) stand in the prime line of enzymatic antioxidant defense in nearly all eukaryotic cells exposed to oxygen, catalyzing the breakdown of the superoxide anionic radical to O(2) and H(2)O(2). Overproduction of superoxide correlates with numerous pathophysiological conditions, and although the native enzyme can be used as a therapeutic agent in superoxide-associated conditions, synthetic low molecular weight mimetics are preferred in terms of cost, administration mode, and bioavailability. In this study we make use of the model eukaryote Saccharomyces cerevisiae to investigate the SOD-mimetic action of a mononuclear mixed-ligand copper(II) complex, [CuCl(acac)(tmed)] (where acac is acetylacetonate anion and tmed is N,N,N',N'-tetramethylethylenediamine). Taking advantage of an easily reproducible phenotype of yeast cells which lack Cu-Zn SOD (Sod1p), we found that the compound could act either as a superoxide scavenger in the absence of native Sod1p or as a Sod1p modulator which behaved differently under various genetic backgrounds. 相似文献
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Ofiteru AM Ruta LL Rotaru C Dumitru I Ene CD Neagoe A Farcasanu IC 《Applied microbiology and biotechnology》2012,94(2):425-435
Pho84p, the protein responsible for the high-affinity uptake and transport of inorganic phosphate across the plasma membrane,
is also involved in the low-affinity uptake of heavy metals in the Saccharomyces cerevisiae cells. In the present study, the effect of PHO84 overexpression upon the heavy metal accumulation by yeast cells was investigated. As PHO84 overexpression triggered the Ire1p-dependent unfolded protein response, abundant plasma membrane Pho84p could be achieved
only in ire1Δ cells. Under environmental surplus, PHO84 overexpression augmented the metal accumulation by the wild type, accumulation that was exacerbated by the IRE1 deletion. The pmr1Δ cells, lacking the gene that encodes the P-type ATPase ion pump that transports Ca2+ and Mn2+ into the Golgi, hyperaccumulated Mn2+ even from normal medium when overexpressing PHO84, a phenotype which is rather restricted to metal-hyperaccumulating plants. 相似文献
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Federico Capra Diego Abalos Stefania Codruta Maris Federico Ardenti Michela Lommi Vincenzo Tabaglio Andrea Fiorini 《Global Change Biology Bioenergy》2023,15(7):867-885
Digestate, a by-product of biogas production, is widely recognized as a promising renewable nitrogen (N) source with high potential to replace synthetic fertilizers. Yet, inefficient digestate use can lead to pollutant N losses as ammonia (NH3) volatilization, nitrous oxide (N2O) emissions and nitrate () leaching. Cover crops (CCs) may reduce some of these losses and recycle the N back into the soil after incorporation, but the effect on the N balance depends on the CC species. In a one-year field study, we tested two application methods (i.e., surface broadcasting, BDC; and shallow injection, INJ) of the liquid fraction of separated co-digested cattle slurry (digestate liquid fraction [DLF]), combined with different winter cover crop (CC) options (i.e., rye, white mustard or bare fallow), as starter fertilizer for maize. Later, side-dressing with urea was required to fulfil maize N-requirements. We tested treatment effects on yield, N-uptake, N-use efficiency parameters, and N-losses in the form of N2O emissions and leaching. CC development and biomass production were strongly affected by their contrasting frost tolerance, with spring-regrowth for rye, while mustard was winter killed. After the CCs, injection of DLF increased N2O emissions significantly compared with BDC (emission factor of 2.69% vs. 1.66%). Nitrous oxide emissions accounted for a small part (11%–13%) of the overall yield-scaled N losses (0.46–0.97 kg N Mg grain−1). The adoption of CCs reduced fall leaching, being 51% and 64% lower for mustard and rye than under bare soil. In addition, rye reduced leaching during spring and summer after termination by promoting N immobilization, thus leading to −57% lower annual leaching losses compared with mustard. DLF application method modified N-loss pathways, but not the cumulative yield-scaled N losses. Overall, these insights contribute to inform an evidence-based design of cropping systems in which nutrients are recycled more efficiently. 相似文献
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