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

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

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

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 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

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 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 /1L Filter sterilise - 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 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.

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 50 kDa 20 kDa L 3 hrs 5 hrs 8 hrs Gel courtesy of Patricia Zunszain

Results: BL 21 (DE3) pLysS 3Cpro 50 kDa 20 kDa L 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): 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.