Trevor Sweeney Curry Group

1. 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. 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): 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

5. Auto-induction Lactose Glycerol Glucose Extracellular Intracellular Glucose » Early energy source » Repression Glycerol » Late energy source Lactose » Induction Lactose cAMP Lactose to Allolactose Lactose Permease LacI CRP β-Gal X lacI Prom lacZlacY 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 20-24 hrs • Harvest cells by centrifugation • Typical cell densities OD600 5-6

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 - 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

11. Antibiotics *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) 50 kDa 3Cpro 20 kDa L 3 hrs 5 hrs 8 hrs Gel courtesy of Patricia Zunszain

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