Figure 2. Expression of TSOL18-GST in E. coli. A, B, comparison of E. coli cultures expressing TSOL18-GST that were incubated at different temperatures and induced using various concentrations of isopropyl β-D thiogalactopyranoside (IPTG). SDS-PAGE of insoluble proteins (A) and soluble proteins (B) from bacterial lysates. 1–4, samples from cultures incubated at 30°C and induced with 0.2 mM, 0.5 mM, 1 mM and 2 mM IPTG respectively. 5, sample from culture induced with 0.2 mM IPTG and incubated at 37°C. 6, no IPTG. M, protein markers (kDa). Arrows denote position of TSOL18-GST. C, analysis of samples from an optimized fermentor culture containing E. coli expressing TSOL18-GST. A 10 L Biostat B Plus fermentor (Sartorius) was used in the fermentation and expression was induced with 0.2 mM IPTG when the OD_595nm reached 20 units (0 h in [C]). The culture was maintained at pH 7.5 and 50% dissolved oxygen (DO) concentration. The DO concentration was maintained using a cascade of agitation between 200–800 rpm and air supplied at 0.4–20 l/min via a Biostat Bplus micro-DCU. Pure oxygen was blended into the air at high cell densities and was regulated via the control unit. Temperature was set at 37°C and reduced to 30°C at the initiation of induction with IPTG. Super broth (35 g soy peptone, 20 g yeast extract, 5 g NaCl and 100 mg ampicillin per liter) was used in the fermentation. Super broth feed (containing 20% glycerol) was added at 5 ml/min when OD_595nm reached 3 units. Half hourly samples were removed from the fermentation and used to determine OD_595nm, cell wet weight, % TSOL18 in total soluble proteins from lysed E.coli and TSOL18 expression yields (C). The percentage of TSOL18-GST in total soluble E.coli proteins was determined by scanning densitometry (Molecular Dynamics) of culture samples separated by SDS-PAGE, following removal of medium by centrifugation, lysis by sonication in phosphate buffered saline and removal of insoluble solids by centrifugation at 10,000 g. TSOL18 expression yields were determined from the soluble protein fractions of culture samples by scanning densitometry and comparison to standards of known mass on SDS-PAGE.