Presentation on theme: "Trevor Sweeney Curry Group Auto-induction for over expression in E. coli Centre for Structural Biology Techniques Workshop on Cloning and Expression."— Presentation transcript:
Trevor Sweeney Curry Group Auto-induction for over expression in E. coli Centre for Structural Biology Techniques Workshop on Cloning and Expression
Protein expression in E. coli Protein coding sequence cloned into plasmid under the control of T7 promoter Plasmid used to transform E. coli that possess an inducible T7 polymerase Little expression in absence of induction After induction most protein synthesis directed towards target protein
IPTG Induction lacI Prom lacO ATG STOP No T7 or Target Protein expressed lacI Prom lacO ATG STOP - IPTG + IPTG T7 and Target protein expressed
Auto-induction Method described by Studier - Studier FW (2005) Protein Expr. Purif. 41(1): Based on ability of certain media to induce protein expression in E. coli when cells reach saturation Result of the different metabolism states of the bacteria Complete study on what components of the media are necessary for auto-induction
Auto-induction cAMP CRP LacI Lactose Permease β-Gal Prom lacZ lacY Lactose to Allolactose Glucose » Early energy source » Repression Glycerol » Late energy source Lactose » Induction Extracellular Intracellular Glucose Bacterial Genome Lactose lacI X Glycerol
Studiers main conclusions Auto-induction is a result of lactose in the media Glucose prevents induction by lactose Auto-induction can be regulated by adjusting glucose/lactose levels in media
General Procedure Transform E. coli with desired plasmid Inoculate 1 L of culture media with a single colony Incubate with shaking for hrs Harvest cells by centrifugation Typical cell densities OD
Cell types BL21 (DE3) - T7 polymerase present in chromosome Compatible with B834 (DE3), C41 (DE3) Cell types expressing lysozyme (e.g. pLysS) are not recommended Suitable for expression of labelled protein
Media Autoclave /1L - Phosphate Buffer (pH 7.2) 6g Na 2 HPO 4 /3g KH 2 PO 4 - Tryptone 20 g - Yeast Extract 5 g - NaCl 5 g Filter sterilise - 60 % v/v Glycerol 10 ml - 10 % w/v Glucose 5 ml - 8 % w/v Lactose 25 ml
Antibiotics Antibiotic Final conc. µg/ml Kanamycin*100 Ampicillin50 Chloramphenicol35 *High phosphate induces Kanamycin resistance, 100 µg/ml is sufficient when using media described above.
Method Expression from a single colony usually works - but not always! Test small scale cultures for induction Save aliquot 1 hr after start of small culture- store at 4 °C Take sample after 5 hrs and again 3 hrs later Compare on gel - use best inducing cells for large scale
Results: BL 21 (DE3) 3C pro L 3 hrs 5 hrs 8 hrs 50 kDa 20 kDa Gel courtesy of Patricia Zunszain
Results: BL 21 (DE3) pLysS L 3 hrs 5 hrs 8 hrs 3C pro 50 kDa 20 kDa Gel courtesy of Patricia Zunszain
Benefits over IPTG induction No need to monitor OD 600 Can run multiple inductions in parallel Final OD 600 is much greater than with IPTG induction (LB/IPTG ~ 1.8, Auto-induction ~ 5) Increased protein yields Protein expressed while you sleep!
Commercial media Advertising feature - Grabski A et al. (2005) Nat. Meth. 2, 233 – 235. Pre-prepared auto-induction media - powder form, just add water - Microwave to sterilise Demonstrate 2-fold higher protein yield with twice the cell density compared to IPTG induction
Potential Problems Occasionally protein expressed in this way has been degraded Returned to regular IPTG induction for these targets
Papers Studier FW (2005), Protein Production by Auto-Induction in High-Density Shaking Cultures. Protein Expr. Purif. 41(1): 207–234. Grabski A, Mehler M, Drott D (2005), The Overnight Express Autoinduction System: High-density cell growth and protein expression while you sleep. Nature Methods 2, 233 – 235.