Auto-induction for over expression in E. coli

Autoinduction 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 c[.]

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

• 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

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

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

- 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

µg/ml

• 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)

3C pro

50 kDa

20 kDa

Results: BL 21 (DE3) pLysS

3C pro

50 kDa

20 kDa

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

al (2005) Nat Meth 2, 233 – 235.

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

• 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.