The resulting PCR products were digested with PciI and ligated to the PciI digested vector pTH1. The resulting vectors were named pTH1-tkt C (Bme) and pTH1-tkt P (Bme), respectively. Crude cell extracts were prepared based on the protocol described elsewhere [20]. B. methanolicus cells were grown in SOB medium with 0.25 mM

sucrose to stationary phase (OD600, 2.5 to 3.3). Gene selleck chemicals expression was induced by addition of 200 mM methanol at inoculation. 20 ml of the cell culture was harvested by centrifugation (4000 × g, 10 min, 4°C), washed in 50 mM potassium phosphate buffer (pH 7.5) and stored at -20°C. The cells were disrupted by sonication described [29]. Cell debris was removed Captisol clinical trial by centrifugation (14,000 x g, 1 h, 4°C) and the supernatant was collected as crude extract. TKT activity was measured according to assay II. Purification molecular mass determination of TKT proteins For protein production with E. coli BL21 (DE3) [61], tkt P and tkt C were amplified by PCR using the primers Nepicastat price tkt_C-Xho-fw and tkt_C-Xho-rv and tkt_P-Xho-fw and tkt_P-Xho-rv (Table 3). The resulting PCR products were ligated, after restriction with XhoI, into XhoI restricted

pET16b (Novagen, Madison, Wisconsin, USA), resulting in pET16b-tkt C and pET16b-tkt P . The pET16b vector allows the production of an N-terminal decahistidine tagged TKT in E. coli BL21 (DE3). Protein production and purification was performed as described previously [62]. Both enzymes were purified to homogenity. After purification, the His-tag was cleaved by factor Xa (Novagen, San Diego) according to the manufacturer’s recommendations and buffered in 20 mM Tricine, pH 7.7. The protein purification was analyzed by 12% SDS-PAGE [63]. Protein concentration was measured according the method of Bradford using the Bio-Rad Protein-Assay Dimethyl sulfoxide with BSA as standard. The tetrameric structures of the TKT proteins were determined by gel filtration as described previously [62] using 1 mg TKT dissolved in 2 ml of 20 mM Tris–HCl, pH 7.5. Enzyme assays for

the purified TKT proteins The TKT activity in the direction of S7P + GAP from R5P + Xu5P was done by Assay I, a modified version of a previously described assay [31] using the auxiliary enzymes triose-phosphate isomerase (TPI) and glycerol 3-phosphate dehydrogenase (GPD) from rabbit muscle. The oxidation of NADH was followed setting 1 pmol NADH oxidized equivalent to 1 pmol X5-P consumed. The standard reaction mixture (final volume 1 ml) contained 50 mM Tris–HCl buffer (pH 7.5), 0.25 mM NADH, 2 mM Mn2Cl, 0.4 U/ml TPI, 0.7 U/ml glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and purified TKT protein which was preheated for 3 min at 50°C. NADH reduction (ϵ340nm = 6.22 mM–1 cm–1) was followed at 340 nm on a Shimadzu UV1700 spectrophotometer. The reaction was initiated by the addition of R5-P or X5-P, respectively (final concentration varied between 0.05 – 10 mM).