Auto-induction for over expression in E. coli

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): 207-234.
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
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
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
General Procedure
Transform E. coli with desired plasmid
Inoculate 1 L of culture media with a single colony
Incubate with shaking for 20-24 hrs
Harvest cells by centrifugation
Typical cell densities OD600 5-6
Vectors
pET vectors:
T7 promoter
Iac operator
lacI
Antibiotic
resistance
T7 Promotor
lacO
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 Na2HPO4/3g KH2PO4
- 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
*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)

3Cpro
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
3Cpro
50 kDa
20 kDa
Gel courtesy of Patricia Zunszain
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!
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.