PCR verified expression constructs were transformed into either B834 (DE3) or Rosetta(DE3)LysS E. coli (Novagen) in 96-tube format as described for OmniMaxII transformations with 1% w/v Glucose replacing the X-Gal and IPTG reagents in the LB agar. All plates and subsequent media used for the culture of the Rosetta(DE3) LysS cells are identical to those described for B834(DE3) cells but also supplemented with 35 µg/ml chloramphenicol to maintain the pRareLysS plasmid. Plates were incubated for 18 h at 37°C before individual colonies were used to inoculate, 500 µl GS96 (Bio101, QBioGene, Cambridge, UK) supplemented with 0.05% v/v glycerol, 1% w/v glucose and 50 µg/ml carbenicillin in 96-well deep-well plates. The plates were sealed with gas-permeable adhesive seals and shaken at 225 r.p.m. at 37°C for 18 h. For IPTG induction of expression, 50 µl of each overnight culture was then used to inoculate (in four 24-well deep-well plates) 2.5 ml of GS96 supplemented with 50 µg/ml carbenicillin. The diluted cultures were grown at 37°C with shaking at 225 r.p.m., for 3 h before reducing the temperature to 20°C, addition of ITPG to a final concentration of 0.5 mM and shaking for a further 18 h at 20°C. For auto-induction of expression, 50 µl of each overnight culture was used to inoculate (in four 24-well deep-well plates) 2.5 ml of Overnight Express™ Instant TB media (Novagen) supplemented with 50 µg/ml carbenicillin. The diluted cultures were grown at 37°C with shaking at 225 r.p.m., for 3 h before reducing the temperature to 25°C and shaking for a further 24 h at 25°C.
A 1.5 ml aliquot of culture from each well was then transferred to a 2 ml 96-well deep-well plate using a Theonyx robot (Aviso Gmbh, Gera, Germany) and harvested by centrifugation at 6000 g for 10 min at 4°C before decanting of the waste media. Pelleted cells were frozen at −80°C for at least 30 min prior to screening for soluble His6-tag protein expression using either the Theonyx or BR8000 robotic platforms with standard Qiagen Ni-NTA magnetic bead protocols (as per manufacturer's instructions). Proteins purified by elution from the Ni-NTA beads were analysed on SDS-PAGE gels (Criterion™ 10–20% gradient gels—Biorad, Hemel Hempstead, UK or InVitrogen NuPAGE™ Novex 10% Bis-Tris Midi gels with MES buffer system) and visualized with SafeStain™ (InVitrogen). Scale-up and purification of proteins from E. coli were carried out as described earlier (11 (link)).
A 1.5 ml aliquot of culture from each well was then transferred to a 2 ml 96-well deep-well plate using a Theonyx robot (Aviso Gmbh, Gera, Germany) and harvested by centrifugation at 6000 g for 10 min at 4°C before decanting of the waste media. Pelleted cells were frozen at −80°C for at least 30 min prior to screening for soluble His6-tag protein expression using either the Theonyx or BR8000 robotic platforms with standard Qiagen Ni-NTA magnetic bead protocols (as per manufacturer's instructions). Proteins purified by elution from the Ni-NTA beads were analysed on SDS-PAGE gels (Criterion™ 10–20% gradient gels—Biorad, Hemel Hempstead, UK or InVitrogen NuPAGE™ Novex 10% Bis-Tris Midi gels with MES buffer system) and visualized with SafeStain™ (InVitrogen). Scale-up and purification of proteins from E. coli were carried out as described earlier (11 (link)).
