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Fig. 3. In vitro and in vivo activity of Cgs CR I mutants

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1 Fig. 3. In vitro and in vivo activity of Cgs CR I mutants
Fig. 3. In vitro and in vivo activity of Cgs CR I mutants. In vitro activity was determined using permeabilized cells. Incorporation of [<sup>14</sup>C]glucose into Cgs protein (TCA-insoluble fraction) (A) and cyclic glucan (water-soluble fraction) (C) was quantified, as described in Materials and Methods. The bar graph data are the means and standard deviation for three separate enzyme activity determinations. The percentage of activity for the mutants with very low activity is indicated over the corresponding bar graph. TCA-insoluble fractions were also subjected to Coomassie Blue-staining SDS–PAGE (B, upper panel), and radioactivity was detected by fluorography (B, lower panel). As a measure of in vivo activity, cyclic glucans were extracted from cell pellets by the ethanol method and analyzed by TLC (D) (similar results were obtained in three different experiments). The position of molecular mass standard (in kilodaltons) is indicated on the left. The arrows on the right indicate the position of Cgs protein. In vitro activity of the mutants was determined at 28°C incubating 5, 10, and 20 min. Only the values for the point of 20 min are shown. WT, S19Δcgs strain carrying the plasmid pBA22; cgs, S19Δcgs strain. * and **, migration of anionic and neutral Brucella abortus cyclic β-1,2-glucan, respectively (Roset, M.S., Ciocchini, A.E., Ugalde, R.A., and Iñón de Iannino, N., in preparation). From: Identification of active site residues of the inverting glycosyltransferase Cgs required for the synthesis of cyclic β-1,2-glucan, a Brucella abortus virulence factor Glycobiology. 2006;16(7): doi: /glycob/cwj113 Glycobiology | © The Author Published by Oxford University Press. All rights reserved. For permissions, please 1


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