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.