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61.
Expression of recombinant galactose oxidase by Pichia pastoris 总被引:5,自引:0,他引:5
Galactose oxidase catalyzes the oxidation of a variety of primary alcohols, producing hydrogen peroxide as a product. Among hexose sugars, the enzyme exhibits a high degree of specificity for the C6-hydroxyl of galactose and its derivatives, underlying a number of important bioanalytical applications. Galactose oxidase cDNA has been cloned for expression in Pichia pastoris both as the full-length native sequence and as a fusion with the glucoamylase signal peptide. Expression of the full-length native sequence results in a mixture of partly processed and mature galactose oxidase. In contrast, the fusion construct directs efficient secretion of correctly processed galactose oxidase in high-density, methanol-induced fermentation. Culture conditions (including induction temperature and pH) have been optimized to improve the quality and yield (500 mg/L) of recombinant enzyme. Lowering the temperature from 30 to 25 degrees C during the methanol induction phase results in a fourfold increase in yield. A simple two-step purification and one-step activation produce highly active galactose oxidase suitable for a wide range of biomedical and bioanalytical applications. 相似文献
62.
Marker-assisted selection using ridge regression 总被引:4,自引:0,他引:4
In cross between inbred lines, linear regression can be used to estimate the correlation of markers with a trait of interest; these marker effects then allow marker assisted selection (MAS) for quantitative traits. Usually a subset of markers to include in the model must be selected: no completely satisfactory method of doing this exists. We show that replacing this selection of markers by ridge regression can improve the mean response to selection and reduce the variability of selection response. 相似文献
63.
64.
G. W. Tinney P. E. Hatcher P. G. Ayres N. D. Paul J. B. Whittaker 《Entomologia Experimentalis et Applicata》1998,88(2):137-145
The effects of sublethal doses of deltamethrin and propoxur, applied topically at LD10, LD30 and LD50 on German cockroaches, were studied by reciprocal crossing. Male and female longevities decreased curvilinearly with increasing sublethal doses of deltamethrin, and decreased linearly with increasing sublethal doses of propoxur. Fecundity of females treated with deltamethrin and propoxur was reduced with increasing sublethal doses of both insecticides. Oothecal production, oothecal hatchability and nymphal production also declined with increasing doses of deltamethrin and propoxur. Preoviposition and incubation periods were not affected by sublethal doses of deltamethrin and propoxur, although some significant differences were observed at certain oothecal numbers. Two-way analysis of variance indicated that only treated females showed an effect on oothecal production while oothecal hatch and nymphal production were governed by both treated females and males. Insecticide susceptibility tests on the progeny of parents treated with sublethal doses demonstrated that these doses did not increase insecticidal tolerance in the F1 generation. 相似文献
65.
Sara B.-M. Whittaker Ruth Boetzel Colin MacDonald Lu-Yun Lian Ansgar J. Pommer Ann Reilly Richard James Colin Kleanthous Geoffrey R. Moore 《Journal of biomolecular NMR》1998,12(1):145-159
The cytotoxic activity of the secreted bacterial toxin colicin E9 is due to a non-specific DNase housed in the C-terminus of the protein. Double-resonance and triple-resonance NMR studies of the 134-amino acid15 N- and 13C/15N-labelled DNase domain are presented. Extensive conformational heterogeneity was evident from the presence of far more resonances than expected based on the amino acid sequence of the DNase, and from the appearance of chemical exchange cross-peaks in TOCSY and NOESY spectra. EXSY spectra were recorded to confirm that slow chemical exchange was occurring. Unambiguous sequence-specific resonance assignments are presented for one region of the protein, Pro65-Asn72, which exists in two slowly exchanging conformers based on the identification of chemical exchange cross-peaks in 3D 1H-1H-15N EXSY-HSQC, NOESY-HSQC and TOCSY-HSQC spectra, together with C and C chemical shifts measured in triple-resonance spectra and sequential NH NOEs. The rates of conformational exchange for backbone amide resonances in this stretch of amino acids, and for the indole NH of either Trp22 or Trp58, were determined from the intensity variation of the appropriate diagonal and chemical exchange cross-peaks recorded in 3D1 H-1H-15N NOESY-HSQC spectra. The data fitted a model in which this region of the DNase has two conformers, NA and NB, which interchange at 15 °C with a forward rate constant of 1.61 ± 0.5 s-1 and a backward rate constant of 1.05 ± 0.5 s-1. Demonstration of this conformational equilibrium has led to a reappraisal of a previously proposed kinetic scheme describing the interaction of E9 DNase with immunity proteins [Wallis et al. (1995) Biochemistry, 34, 13743–13750 and 13751–13759]. The revised scheme is consistent with the specific inhibitor protein for the E9 DNase, Im9, associating with both the NA and NB conformers of the DNase and with binding only to the NB conformer detected because the rate of dissociation of the complex of Im9 and the NA conformer, NAI, is extremely rapid. In this model stoichiometric amounts of Im9 convert, the E9 DNase is converted wholly into the NBI form. The possibility that cis–trans isomerisation of peptide bonds preceding proline residues is the cause of the conformational heterogeneity is discussed. E9 DNase contains 10 prolines, with two bracketing the stretch of amino acids that have allowed the NA NB interconversion to be identified, Pro65 and Pro73. The model assumes that one or both of these can exist in either the cis or trans form with strong Im9 binding possible to only one form. 相似文献
66.
William H. Schlesinger L.A. Bruijnzeel Mark B. Bush Emily M. Klein Kimberly A. Mace Jane A. Raikes R.J. Whittaker 《Biogeochemistry》1998,40(1):37-55
This study examined the accumulation of organic carbon (C) and fractions ofsoil phosphorus (P) in soils developing in volcanic ash deposited in the1883 eruption of Krakatau. Organic C has accumulated at rates of 45 to 127g/m2/yr during 110 years of soil development, resulting inprofiles with as much as 14 kgC/m2. Most soil P is found inthe HCl-extractable forms, representing apatite. A loss of HCl-extractableP from the surface horizons is associated with a marked accumulation ofNaOH-extractable organic P bound to Al. A bioassay with hill rice suggeststhat P is limiting to plant growth in these soils, perhaps as a result ofthe rapid accumulation of P in organic forms. 相似文献
67.
Stephen P. East Samantha Bamford Matthias G.A. Dietz Christian Eickmeier Adam Flegg Boris Ferger Mark J. Gemkow Ralf Heilker Bastian Hengerer Adrian Kotey Pui Loke Gerhard Schänzle Hans-Dieter Schubert John Scott Mark Whittaker Mildred Williams Przemyslaw Zawadzki Kai Gerlach 《Bioorganic & medicinal chemistry letters》2010,20(16):4901-4905
A high-throughput screening campaign identified 4-((E)-styryl)-pyrimidin-2-ylamine (11) as a positive allosteric modulator of the metabotropic glutamate (mGlu) receptor subtype 4. An evaluation of the structure–activity relationships (SAR) of 11 is described and the efficacy of this compound in a haloperidol-induced catalepsy rat model following oral administration is presented. 相似文献
68.
Jennifer MP Woo Zhuofeng Lin Mohamad Navab Casey Van Dyck Yvette Trejo-Lopez Krystal MT Woo Hongyun Li Lawrence W Castellani Xuping Wang Noriko Iikuni Ornella J Rullo Hui Wu Antonio La Cava Alan M Fogelman Aldons J Lusis Betty P Tsao 《Arthritis research & therapy》2010,12(3):R93
Introduction
The purpose of this study was to evaluate the effects of L-4F, an apolipoprotein A-1 mimetic peptide, alone or with pravastatin, in apoE-/-Fas-/-C57BL/6 mice that spontaneously develop immunoglobulin G (IgG) autoantibodies, glomerulonephritis, osteopenia, and atherosclerotic lesions on a normal chow diet.Methods
Female mice, starting at eight to nine weeks of age, were treated for 27 weeks with 1) pravastatin, 2) L-4F, 3) L-4F plus pravastatin, or 4) vehicle control, followed by disease phenotype assessment.Results
In preliminary studies, dysfunctional, proinflammatory high-density lipoproteins (piHDL) were decreased six hours after a single L-4F, but not scrambled L-4F, injection in eight- to nine-week old mice. After 35 weeks, L-4F-treated mice, in the absence/presence of pravastatin, had significantly smaller lymph nodes and glomerular tufts (PL, LP < 0.05), lower serum levels of IgG antibodies to double stranded DNA (dsDNA) (PL < 0.05) and oxidized phospholipids (oxPLs) (PL, LP < 0.005), and elevated total and vertebral bone mineral density (PL, LP < 0.01) compared to vehicle controls. Although all treatment groups presented larger aortic root lesions compared to vehicle controls, enlarged atheromas in combination treatment mice had significantly less infiltrated CD68+ macrophages (PLP < 0.01), significantly increased mean α-actin stained area (PLP < 0.05), and significantly lower levels of circulating markers for atherosclerosis progression, CCL19 (PL, LP < 0.0005) and VCAM-1 (PL < 0.0002).Conclusions
L-4F treatment, alone or with pravastatin, significantly reduced IgG anti-dsDNA and IgG anti-oxPLs, proteinuria, glomerulonephritis, and osteopenia in a murine lupus model of accelerated atherosclerosis. Despite enlarged aortic lesions, increased smooth muscle content, decreased macrophage infiltration, and decreased pro-atherogenic chemokines in L-4F plus pravastatin treated mice suggest protective mechanisms not only on lupus-like disease, but also on potential plaque remodeling in a murine model of systemic lupus erythematosus (SLE) and accelerated atherosclerosis. 相似文献69.
70.
Pan HY Whittaker MM Bouveret R Berna A Bernier F Whittaker JW 《Biochemical and biophysical research communications》2007,356(4):925-929
High-level secretory expression of wheat (Triticum aestivum) germin/oxalate oxidase was achieved in Pichia pastoris fermentation cultures as an alpha-mating factor signal peptide fusion, based on the native wheat cDNA coding sequence. The oxalate oxidase activity of the recombinant enzyme is substantially increased (7-fold) by treatment with sodium periodate, followed by ascorbate reduction. Using these methods, approximately 1 g (4x10(4) U) of purified, activated enzyme was obtained following eight days of induction of a high density Pichia fermentation culture, demonstrating suitability for large-scale production of oxalate oxidase for biotechnological applications. Characterization of the recombinant protein shows that it is glycosylated, with N-linked glycan attached at Asn47. For potential biomedical applications, a nonglycosylated (S49A) variant was also prepared which retains essentially full enzyme activity, but exhibits altered protein-protein interactions. 相似文献