Influence of the Media Composition on Behavior of pET Expression Systems
Simona Jevševar1*, Jernej Palčič3, Špela Jalen2, Aleksander Pavko3
1 Lek Pharmaceuticals d.d., a Sandoz company,
Biopharmaceuticals, New Technologies Department,Laboratory location: Hajdrihova
19, SI-1000 Ljubljana, Slovenia.
Phone:++386 1 4760 200,
Fax: ++386 1 4760 300,
2 National Institute of Chemistry, Department of Biosynthesis and Biotransformation, Hajdrihova 19, SI-1000 Ljubljana, Slovenia
3 Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, SI-1000 Ljubljana, Slovenia
† Dedicated to the memory of dr. Viktor Menart, Assistant Professor
pET expression systems (Novagen) are the strongest tool available for production of recombinant proteins in bacteria E. coli and have been widely used for many years. It is relatively difficult to control them due to their efficiency enabling formation of large amounts of recombinant proteins. Our work was focused on the influence of the media composition on the behavior of the pET3a expression system and we tried to select appropriate medium for inoculum preparation as well as appropriate production medium. We found out that media without glucose trigger unexpectedly high activity of lacUV5 promoter. Accumulation level of 15% of human granulocyte colony-stimulating factor (hG-CSF) in total cellular proteins was obtained in LBP medium (modified Luria-Bertani) without addition of IPTG inducer. Glucose addition into medium for inoculum preparation successfully represses expression of recombinant protein during inoculum preparation phase. Optimal optical density for high quality of inoculum is around OD600nm = 4,0, when culture is in the middle of exponential growth phase. Presence of glucose is required also in production medium. GYSP medium containing glucose, enables by 25% higher recombinant protein accumulation level than LBP medium without glucose. In contrast to LBP medium it enables comparably high recombinant protein accumulation level also without addition of antibiotic into the production medium.
Keywords: E. coli, T7 promoter, media composition, catabolic repression, hG-CSF, IPTG