Presentation on theme: "Trevor Sweeney Curry Group"— Presentation transcript:
1Trevor Sweeney Curry Group Auto-induction forover expression in E. coliCentre for Structural BiologyTechniques Workshop on Cloning and ExpressionTrevor SweeneyCurry Group
2Protein expression in E. coli Protein coding sequence cloned into plasmid under the control of T7 promoterPlasmid used to transform E. coli that possess an inducible T7 polymeraseLittle expression in absence of inductionAfter induction most protein synthesis directed towards target protein
3Target Protein expressed Target protein expressed IPTG Induction- IPTGlacI Prom lacO ATG STOPNo T7 orTarget Protein expressed+ IPTGlacI Prom lacO ATG STOPT7 andTarget protein expressed
4Auto-inductionMethod 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 saturationResult of the different metabolism states of the bacteriaComplete study on what components of the media are necessary for auto-induction
5Auto-induction X Extracellular Intracellular Glucose LactoseGlycerolExtracellularIntracellularGlucose» Early energy source» RepressionGlycerol» Late energy sourceLactose» InductioncAMPLactoseLactose toAllolactoseLactosePermeaseCRPLacIβ-GalXlacIProm lacZ lacYBacterial Genome
6Studier’s main conclusions Auto-induction is a result of lactose in the mediaGlucose prevents induction by lactoseAuto-induction can be regulated by adjusting glucose/lactose levels in media
7General Procedure Transform E. coli with desired plasmid Inoculate 1 L of culture media with a single colonyIncubate with shaking for hrsHarvest cells by centrifugationTypical cell densities OD
9Cell types BL21 (DE3) - T7 polymerase present in chromosome Compatible with B834 (DE3), C41 (DE3)Cell types expressing lysozyme (e.g. pLysS) are not recommendedSuitable for expression of labelled protein
11Antibiotics Antibiotic Final conc. µg/ml Kanamycin* 100 Ampicillin 50 Chloramphenicol35*High phosphate induces Kanamycin resistance,100 µg/ml is sufficient when using media described above.
12Method Expression from a single colony usually works - but not always! Test small scale cultures for inductionSave aliquot 1 hr after start of small culture- store at 4 °CTake sample after 5 hrs and again 3 hrs laterCompare on gel - use best inducing cells for large scale
13Results: BL 21 (DE3) 3Cpro 50 kDa 20 kDa L 3 hrs 5 hrs 8 hrs Gel courtesy of Patricia Zunszain
14Results: BL 21 (DE3) pLysS 3Cpro 50 kDa 20 kDa L 3 hrs 5 hrs 8 hrs Gel courtesy of Patricia Zunszain
15Benefits over IPTG induction No need to monitor OD600Can run multiple inductions in parallelFinal OD600 is much greater than with IPTG induction (LB/IPTG ~ 1.8, Auto-induction ~ 5)Increased protein yieldsProtein expressed while you sleep!
16Commercial mediaAdvertising feature Grabski A et al. (2005) Nat. Meth. 2, 233 – 235.Pre-prepared auto-induction media powder form, just add water- Microwave to steriliseDemonstrate 2-fold higher protein yield with twice the cell density compared to IPTG induction
17Potential ProblemsOccasionally protein expressed in this way has been degradedReturned to regular IPTG induction for these targets
18PapersStudier 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.