Bacillusap: Acid phosphatase of Bacillus licheniformis Cryparpgm

Bacillusap: Acid phosphatase of Bacillus licheniformis. Cryparpgm: Phosphoglycerate domain of P5091 datasheet Cryptosporidium parvum. E.colidpgM: Cofactor dependent phosphoglycerate mutase of E. coli. PhoE: Acid phosphatase of Bacillus stearothermophillus. Rv0489: Cofactor dependent phosphoglycerate mutase of M. tuberculosis. Rv2419c: Glucosyl-3-phosphoglycerate phosphatase of M. tuberculosis. Rv3214: Acid phosphatase of M. tuberculosis. Rv3837c: Probable cofactor dependent phosphoglycerate mutase of M. tuberculosis. YDR051pgm: Cofactor dependent phosphoglycerate mutase of Saccharomyces arboricola. Functions of Bacillusap, Cryparpgm

and Rv3837c were predicted with bioinformatics while E.colidpgM, Rv0489, PhoE, Rv2419c, www.selleckchem.com/products/dinaciclib-sch727965.html Rv3214 and YDR051pgm have been experimentally characterized. Cloning and expression of C-His-Rv2135c and C-His-Rv0489 Rv2135c and Rv0489 genes of M. tuberculosis were successfully cloned with

6 histidine codons tagged at the 3′ end. The recombinant proteins were successfully expressed in E. coli BL21(DE3), resulting in appearance of extra protein bands with the sizes of about 27 kDa and 28 kDa in the soluble fraction of the cell lysates on SDS-PAGE. The sizes are PI3K inhibitor in agreement with the amino acid calculated sizes of 25.95 kDa and 28 kDa respectively. C-His-Rv2315c and C-His-Rv0489 were purified to near homogeneity as shown in Figures 2 and 3, in a single step by loading into the cobalt charged resin column and eluting either by an increasing gradient of imidazole or fixed concentration of imidazole. The method resulted in about 40% yield and 2.4 folds increase in specific activity compared to the crude extract for C-His-Rv0489 as shown in Table 1. About 60% yield and 5.6 folds

increase in specific activity compared to the crude extract for C-His-Rv2135c, when assayed at pH 5.8, were obtained as shown in Table 2. Figure 2 12.5% SDS-PAGE of C-His-Rv2135c expressed in E. coli BL21(DE3) with or without induction and its purified form. Lane 1: 5 μl of 10–250 kDa protein ladder (New England Biolabs). Lane 2: 10 μg of crude lysate of E. coli BL21(DE3) without any plasmid. Lane 3: 8.5 μg of crude Hydroxychloroquine chemical structure lysate of E. coli BL21(DE3)-35c before induction with IPTG. Lane 4: 30 μg of crude lysate of BL21(DE3)-35c after induction with 0.4 mM IPTG for 8 hours at 25°C. Lane 5: 4 μg of recombinant C-His-Rv2135c eluted from IMAC column. Figure 3 12.5% SDS-PAGE of C-His-Rv0489 expressed in E. coli BL21(DE3) with or without induction and its purified form. Lane 1: 9 μl of protein ladder (Fermentas SM0431). Lane 2: 15 μg of crude lysate of E. coli BL21(DE3) without any plasmid. Lane 3: 20 μg of crude lysate of E. coli BL21(DE3)-89 before induction with IPTG. Lane 4: 20 μg of crude lysate of BL21(DE3)-89 after induction with 0.03 mM IPTG overnight at 18°C. The arrow indicates the expressed recombinant protein, C-His-Rv0489. Lane 5: 3.5 μg of recombinant C-His-Rv0489 eluted from IMAC column.

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