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Trevor Sweeney Curry Group

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1 Trevor Sweeney Curry Group
Auto-induction for over expression in E. coli Centre for Structural Biology Techniques Workshop on Cloning and Expression Trevor Sweeney Curry Group

2 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

3 Target Protein expressed Target protein expressed
IPTG Induction - IPTG lacI Prom lacO ATG STOP No T7 or Target Protein expressed + IPTG lacI Prom lacO ATG STOP T7 and Target protein expressed

4 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

5 Auto-induction X Extracellular Intracellular Glucose
Lactose Glycerol Extracellular Intracellular Glucose » Early energy source » Repression Glycerol » Late energy source Lactose » Induction cAMP Lactose Lactose to Allolactose Lactose Permease CRP LacI β-Gal X lacI Prom lacZ lacY Bacterial Genome

6 Studier’s 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

7 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

8 Vectors pET vectors: T7 promoter Iac operator lacI Antibiotic
resistance lacO T7 Promotor

9 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

10 Media Autoclave /1L Filter sterilise
- Phosphate Buffer (pH 7.2) 6g Na2HPO4/3g KH2PO4 - Tryptone g - Yeast Extract g - NaCl g Filter sterilise - 60 % v/v Glycerol ml - 10 % w/v Glucose ml - 8 % w/v Lactose ml

11 Antibiotics Antibiotic Final conc. µg/ml Kanamycin* 100 Ampicillin 50
Chloramphenicol 35 *High phosphate induces Kanamycin resistance, 100 µg/ml is sufficient when using media described above.

12 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

13 Results: BL 21 (DE3) 3Cpro 50 kDa 20 kDa L 3 hrs 5 hrs 8 hrs
Gel courtesy of Patricia Zunszain

14 Results: BL 21 (DE3) pLysS 3Cpro 50 kDa 20 kDa L 3 hrs 5 hrs 8 hrs
Gel courtesy of Patricia Zunszain

15 Benefits over IPTG induction
No need to monitor OD600 Can run multiple inductions in parallel Final OD600 is much greater than with IPTG induction (LB/IPTG ~ 1.8, Auto-induction ~ 5) Increased protein yields Protein expressed while you sleep!

16 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

17 Potential Problems Occasionally protein expressed in this way has been degraded Returned to regular IPTG induction for these targets

18 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.

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