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11.
Expression of the recombinant antibacterial peptide sarcotoxin IA in Escherichia coli cells 总被引:5,自引:0,他引:5
Skosyrev VS Kulesskiy EA Yakhnin AV Temirov YV Vinokurov LM 《Protein expression and purification》2003,28(2):350-356
Sarcotoxin IA is an antibacterial peptide that is secreted by a meat-fly Sarcophaga peregrina larva in response to a hypodermic injury or bacterial infection. This peptide is highly toxic against a broad spectrum of both Gram-positive and Gram-negative bacteria and lethal to microbes even at nanomolar concentrations. However, research needs as well as its potential use in medicine require substantial amounts of highly purified sarcotoxin. Because heterologous expression systems proved to be inefficient due to sarcotoxin sensitivity to intracellular proteases, here we propose the biosynthesis of sarcotoxin precursors in Escherichia coli cells that are highly sensitive to the mature peptide. To optimize its biosynthesis, sarcotoxin was translationally fused with proteins highly expressed in E. coli. A fusion partner and the position of sarcotoxin in the chimeric polypeptide were crucial for protecting the sarcotoxin portion of the fusion protein from proteolysis. Released after chemical cleavage of the fusion protein and purified to homogeneity, sarcotoxin displayed antibacterial activity comparable to that previously reported for the natural peptide. 相似文献
12.
Studies on the biotin-binding site of streptavidin. Tryptophan residues involved in the active site. 总被引:3,自引:1,他引:3 下载免费PDF全文
Streptavidin, the non-glycosylated bacterial analogue of the egg-white glycoprotein avidin, was modified with the tryptophan-specific reagent 2-hydroxy-5-nitrobenzyl (Hnb) bromide. As with avidin, complete loss of biotin-binding activity was achieved upon modification of an average of one tryptophan residue per streptavidin subunit. Tryptic peptides obtained from an Hnb-modified streptavidin preparation were fractionated by reversed-phase h.p.l.c., and three major Hnb-containing peptide fractions were isolated. Amino acid and N-terminal sequence analysis revealed that tryptophan residues 92, 108 and 120 are modified and probably comprise part of the biotin-binding site of the streptavidin molecule. Unlike avidin, the modification of lysine residues in streptavidin failed to result in complete loss of biotin-binding activity. The data imply subtle differences in the fine structure of the respective biotin-binding sites of the two proteins. 相似文献
13.
Zlenko Valerii A. Kotikov Ilia V. Troshin Leonid P. 《Plant Cell, Tissue and Organ Culture》2002,70(3):295-299
Petioles from in vitro grown plants of interspecific grapevine hybrids cvs `Bianca', `Podarok Magaracha' and `Intervitis Magaracha' were cultured on solid NN medium supplemented with 2,4-D and BA at various concentrations. The callus developed was cultured in liquid NN medium supplemented with 0.5 mg l–1 BA to induce formation of somatic embryos. Somatic embryos of globular and heart-stage developed in suspensions of `Podarok Magaracha' and `Intervitis Magaracha'. In contrast, `Bianca' did not undergo embryogenesis beyond globular stage. This made it necessary to perform subculture of the suspensions to HTE liquid medium supplemented with 0.2 mg l–1 BA for the development of globular embryos into heart stage. Heart-stage embryos developed into torpedo-stage after subculturing suspensions of all three cultivars to liquid HTE medium supplemented with 0.1 mg l–1 IAA and 30 mg l–1 sodium hummate. Torpedo-stage embryo suspensions were subcultured in liquid HTE medium supplemented with 0.5 mg l–1 BA, 0.5 mg l–1 GA3 and 0.5 mg l–1 GA3 + 0.2 mg l–1 BA. After 12 days of incubation, plantlets were cultured on solid M2MS medium: without growth regulators and with 0.5 mg l–1 BA. Plantlets that developed in liquid HTE media with 0.5 mg l–1 GA3 or 0.5 mg l–1 GA3 + 0.2 mg l–1 BA produced 82–90% shoots on solid M2MS medium with 0.5 mg l–1 BA after 50 days of culture. 相似文献
14.
Shade light interaction with salicylic acid in regulating growth of sun (alpine) and shade (prairie) ecotypes of Stellaria longipes 总被引:1,自引:0,他引:1
Leonid V. Kurepin Linda J. Walton Allison Hayward R. J. Neil Emery David M. Reid C. C. Chinnappa 《Plant Growth Regulation》2012,66(1):1-8
Magnesium (Mg) deficiency in plants is a widespread problem, affecting productivity and quality in agriculture. The mechanism
of Mg deficiency inducing antioxidant enzyme activities has not been elucidated in rice. We examined the relationship among
abscisic acid (ABA), H2O2, and antioxidant enzymes in the leaves of rice seedlings grown under conditions of Mg deficiency. The expression of OsRab16A, an ABA responsive gene, was used to determine the content of ABA. Mg deficiency resulted in increased ABA content in leaves
of rice seedlings. The production of H2O2 was examined by 3,3-diaminobenzidine staining and a colorimetric method. Mg deficiency also induced H2O2 production in leaves, which was blocked by dipehnyleneiodonium chloride (DPI), an NADPH oxidase inhibitor. Tungstate (Tu),
an ABA biosynthesis inhibitor, was effective in reducing Mg deficiency-increased ABA content, as well as Mg deficiency-induced
H2O2 production. Both Tu and DPI were effective in reducing Mg deficiency-induced activities of superoxide dismutase, ascorbate
peroxidase, glutathione reductase, and catalase in the leaves. Mg deficiency-induced ABA accumulation may trigger increased
production of H2O2, which may involve plasma-membrane NADPH oxidase, and, in turn, up-regulates the activities of antioxidant enzymes in leaves
of rice seedlings. 相似文献
15.
16.
Gareth Forde Aviane Flood Latasha Salter Glake Hill Leonid Gorb Jerzy Leszczynski 《Journal of biomolecular structure & dynamics》2013,31(6):811-817
Abstract Methylation of DNA occurs most readily at N(3), N(7), and O(6) of purine bases and N(3) and O(2) of pyrimidines. Methylated bases are continuously formed through endogenous and exogenous mechanisms. The results of a theoretical ab initio study on the methylation of G:C base pair components are reported. The geometries of the local minima were optimized without symmetry restrictions by the gradient procedure at DFT level of theory and were verified by energy second derivative calculations. The standard 6–31G(d) basis set was used. The single-point calculations have been performed at the MP2/6–31G(d,p), MP2/6–31++G(d,p), and MP2/6–311++G(2d,2p) levels of theory. The geometrical parameters, relative stability and counterpoise corrected interaction energies are reported. Also, using a variation-perturbation energy decomposition scheme we have found the vital contributions to the total interaction energy. 相似文献
17.
Sun Y Breydo L Makarava N Yang Q Bocharova OV Baskakov IV 《The Journal of biological chemistry》2007,282(12):9090-9097
Despite the ability of most proteins to form amyloid, very little is know about amyloid fibril structures and the factors that govern their stability. Using amyloid fibrils produced from full-length prion protein (PrP), we describe a reliable approach for determining both site-specific and global conformational stability of the fibrillar form. To measure site-specific stability, we produced six variants of PrP by replacing the residues at positions 88, 98, 127, 144, 196, and 230 with cysteine, labeled the new cysteines with the fluorescent dye acrylodan, and investigated their conformational status within the amyloid form in guanidine hydrochloride-induced denaturation experiments. We found that the fibrils labeled at positions 127, 144, 196, and 230 displayed cooperative unfolding and showed a very high C1/2 value similar to that observed for the global unfolding of the amyloid structure. The unfolding at residue 98 was also cooperative; however, it showed a C1/2 value substantially lower than that of global unfolding, whereas the unfolding of fibrils labeled at residue 88 was non-cooperative. These data illustrate that there are at least two independent cooperative folding domains within the amyloid structure of the full-length PrP. In addition, kinetic experiments revealed only a partial overlap between the region that constituted the fibrillar cross-beta core and the regions that were involved in nucleation. This result illustrates that separate PrP regions accounted for the nucleation and for the formation of the conformationally most stable fibrillar core. 相似文献
18.
19.
Genome-wide analysis of nucleotide-level variation in commonly used Saccharomyces cerevisiae strains
Ten years have passed since the genome of Saccharomyces cerevisiae-more precisely, the S288c strain-was completely sequenced. However, experimental work in yeast is commonly performed using strains that are of unknown genetic relationship to S288c. Here, we characterized the nucleotide-level similarity between S288c and seven commonly used lab strains (A364A, W303, FL100, CEN.PK, summation 1278b, SK1 and BY4716) using 25mer oligonucleotide microarrays that provide complete and redundant coverage of the approximately 12 Mb Saccharomyces cerevisiae genome. Using these data, we assessed the frequency and distribution of nucleotide variation in comparison to the sequenced reference genome. These data allow us to infer the relationships between experimentally important strains of yeast and provide insight for experimental designs that are sensitive to sequence variation. We propose a rational approach for near complete sequencing of strains related to the reference using these data and directed re-sequencing. These data and new visualization tools are accessible online in a new resource: the Yeast SNPs Browser (YSB; http://gbrowse.princeton.edu/cgi-bin/gbrowse/yeast_strains_snps) that is available to all researchers. 相似文献
20.
Anderson C Bartlett SJ Gansner JM Wilson D He L Gitlin JD Kelsh RN Dowden J 《Molecular bioSystems》2007,3(1):51-59
As a result of a chemical genetic screen for modulators of metalloprotease activity, we report that 2-mercaptopyridine-N-oxide induces a conspicuous undulating notochord defect in zebrafish embryos, a phenocopy of the leviathan mutant. The location of the chemically-induced wavy notochord correlated with the timing of application, thus defining a narrow chemical sensitivity window during segmentation stages. Microscopic observations revealed that notochord undulations appeared during the phase of notochord cell vacuolation and notochord elongation. Notochord cells become swollen as well as disorganized, while electron microscopy revealed disrupted organization of collagen fibrils in the surrounding sheath. We demonstrate by assay in zebrafish extracts that 2-mercaptopyridine-N-oxide inhibits lysyl oxidase. Thus, we provide insight into notochord morphogenesis and reveal novel compounds for lysyl oxidase inhibition. Taken together, these data underline the utility of small molecules for elucidating the dynamic mechanisms of early morphogenesis and provide a potential explanation for the recently established role of copper in zebrafish notochord formation. 相似文献