Overexpression 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 . (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).