Figure 1From: Adenylate kinase-independent thiamine triphosphate accumulation under severe energy stress in Escherichia coliOverexpression of E. coli AK induces cellular ThTP accumulation. Overexpression of E. coli AK induces cellular ThTP accumulation. Strain BL21 λDE3 bacteria carrying pET-21a(+) with the E. coli AK cDNA were grown ovenight in LB medium in the presence of ampicillin (1 mg/ml). The cultures were diluted until A600 = 0.6 – 0.8 and the bacteria were grown in the absence or presence of IPTG (1 mM) for 3 h (37°C, 250 rpm). The bacteria were sonicated, centrifuged and the supernatant was used for electrophoresis (A) and ThTP estimation (B). (A) The presence of a band corresponding to AK was checked on a 12% SDS-PAGE followed by Coomassie blue staining (a, lanes 2, 3 without IPTG and lanes 4, 5 with IPTG). Lanes 1 and 6 contain the molecular weight markers (Prestained Protein Ladder, Invitrogen). Overexpessed protein migrated at Mr 25 000 (arrowhead) for a theoretical molecular mass of 23559 based on amino acid composition [15]. (B) Effect of E. coli AK overexpression on the intracellular [ThTP]/[ThDP] ratio. Aliquots were taken at various times for the determination of thiamine derivatives. The results are expressed as mean ± SD for 3 experiments. At the same time ATP synthesizing activity of AK increased by a factor 1000 in cell-free extracts (not shown).Back to article page