Auto-induction for over expression in E.

coli
Centre for Structural Biology Techniques Workshop on Cloning and Expression

Trevor Sweeney Curry Group

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
- IPTG
lacI Prom lacO ATG STOP

No T7 or Target Protein expressed

+ IPTG
lacI Prom lacO ATG STOP

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
Glucose Lactose Glycerol

Extracellular Glucose Early energy source Repression

Intracellular

cAMP Lactose

Glycerol Late energy source

Lactose Induction CRP

Lactose to Allolactose
-Gal

Lactose Permease

LacI

X
lacI
Prom lacZ lacY Bacterial Genome

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 20-24 hrs

Harvest cells by centrifugation Typical cell densities OD600 5-6

Vectors
pET vectors: T7 promoter Iac operator lacI Antibiotic resistance

lacO T7 Promotor

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
- Phosphate Buffer (pH 7.2) - Tryptone - Yeast Extract - NaCl

/1L
6g Na2HPO4/3g KH2PO4 20 g 5g 5g

Filter sterilise
- 60 % v/v Glycerol - 10 % w/v Glucose - 8 % w/v Lactose 10 ml 5 ml 25 ml

Antibiotics
Antibiotic Kanamycin* Ampicillin Chloramphenicol Final conc. g/ml 100 50 35

*High phosphate induces Kanamycin resistance, 100 g/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 culturestore 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)

50 kDa

20 kDa

3Cpro

3 hrs

5 hrs

8 hrs

Gel courtesy of Patricia Zunszain

Results: BL 21 (DE3) pLysS

50 kDa

20 kDa

3Cpro

3 hrs

5 hrs

8 hrs

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): 207234. 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.