TTTAAACCGC3 ID: 823059
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Chemicals were from Sigma-Aldrich except
Chemicals were from Sigma-Aldrich except as noted. HMP-P kinase and yeast thiamin pyrophosphokinase were prepared as previously described (1, 2, 3). Cloning and DNA manipulations followed standard molecular biological techniques. Automated amplified by PCR using the following sets of primers (inserted restriction sites are underlined): ymfB-F 5-ATTAAGGATGCCATATGTTTAAACCGC-3, ymfB-R 5-ACATTGAACTTCAAGCC-3, yajO-F 5- CAGGAGTG GCCATATGCAATACAACC-3, yajO-R 5-TCATCAGGCCTCGAGTCTTATTTAATTCC-3, yjjX-F 5-GTTAATTTTCTGACATATGCTTATGC-3, yjjX-R 5-CGCGGCA GCGTCGCTCGAGGCGCTTAATACACGGC-3, cof-F 5-GTGGAGAAACATATGGCTCGTCTGGCAGCATTTG-3, cof-R 5-TGCCCGGTGCTCGAGThe sequences of the resulting plasmids were confirmed by automated DNA sequencing, and the plasmids were named pBGL165 (). Each plasmid was transformed into was excised from pBGL175 using sites of pET16b (Novagen) to generate pBGL212, which was transformed into B. subtilisyveN gene was similarly cloned into the sites of pET28a and the resulting plasmid, named pBGLC274, was transformed into Overexpression of ymfB, yajO, yjjX, cof, and yveN.E.
coli37°C in 0.5 L of LB (Gibco BRL) sup
coli37°C in 0.5 L of LB (Gibco BRL) supplemented with chloramphenicol (30 g/mL) to = 0.6, then IPTG was added to a final concentration of 1 mM. Cells were harvested 2 hours after induction, the pellet was resuspended in 3 mL of assay buffer (100 mM Tris-HCl, 5 mM MgClSephadex G-25 PD-10 column (Amersham). YmfB, bearing an N-terminal histidine tag (10X), was prepared in a similar manner from BL21(DE3) carrying pBGL212. The manufacturers protocol (Novagen His-Bind Kits manual, Novagen Inc., Madison, WI) and dialyzed overnight against 50 mM Tris-HCl pH 7.5. YveN was prepared similarly from BL21(DE3) carrying pBGLC274, except protein expression was carried out at 20°C for 5 hrs to increase the solubility of the overexpressed protein. Attempts to purify Cof -HMP-P were prepared from HMP (4) and CF(5) by the method of Cornforth and Popjack (6) as described previously (7). Each product was purified from the reaction mixture on a 1.5 cm x 30 cm DEAE Sepharose A-25 (Sigma) column using a linear gradient of 25 mM to 600 mM ammonium bicarbonate over 200 mL. MeO-HMP-PP and MeO-HMP-P were prepared from bacimet
hrin (8) by reaction with HMP-P kinase
hrin (8) by reaction with HMP-P kinase as described previously (4). MeO-TPP was prepared from 2-methoxythiamin (4) by reaction with yeast thiamin pyrophosphokinase (9). MeO-HMP-PP and MeO-TPP were purified from the enzymatic reaction mixtures by HPLC on a Supelcosil LC-18-T column (15 cm x 4.6 mm, 3 m) (Supelco). Isocratic elution at 1 mL/min with 25 mM ammonium acetate pH 7 provided pure MeO-HMP-PP (2.7 min) and pure MeO-TPP (10.2 min) after lyophilization to remove the buffer. 1. Reddick, J. J., Saha, S., Lee, J.-M., Melnick, J. S., Perkins, J., and Begley, T. P. 2. Baker, L.-J., Dorocke, J. A., Harris, R. A., and Trimm, D. E. (2001) ,539-3. Reddick, J. J., Kinsland, C., Nicewonger, R., Christian, T., Downs, D. M., Winkler, 4. Reddick, J. J., Nicewonger, R., and Begley, T. P. (2001) 5. Barone, J. A., Peters, E., and Tieckelmann, H.(1959)6. Popjak, G., Cornforth, J. W., Cornforth, R. H., Ryhage, R., and Goodman, D. S. 7. Reddick, J. J. (2001) Ph.D. thesis. Cornell University, Ithaca, NY. 8. Perandones, F., and Soto, J. L. (1998) 9. Voskoboev, A. I., Chernikevich, I. P., and Ostrovskii, Y. M.(1976)