Valdez-Cruz et al., 2010 Presentation: July 28 th , 2010

1. Production of recombinant proteins in E. coli by the heat inducible expression system based on the phage lambda pJ and/or pRpromotors Valdez-Cruz et al., 2010 Presentation: July 28th, 2010

2. Why this paper • What actually happens inside the cell in response to genetic engineering, not just how we manipulate and alter cell • Can use to predict responses of the cell • Preemptive preparation against negative response • Different induction system

3. Intro: thermo-regulated expression system for recombinant protein expression • Chemical inducers (eg. IPTG): • expensive • toxic • Possible additional controls to remove chemicals (esp . for human use!) Heat- inducible expression system pros: - λpL/pR system relies on a strong and finely regulated promoter - No special media or toxic chem. Inducers - Culture handling and contaminations risks low - Easily scalable (culture volume) - Yield up to 30% recombinant protein (RP)/ total cell protein • Perfection? Systems based on nutrient exhaustion: (eg. Depletion of an a.a.) - starvation affects cell metabolism, synthesis of the recombinant protein - Precise control of induction timing is difficult

4. Cons/Focus of presentation • Heat shock response (HSR) • Overproduction of RP (often in T7 too) -> heat shock like response, stringent response and a metabolic burden to the cells • Both HSR and RP overproduction-> converge on activation of genes coding for chaperones and proteases (sigma32 regulon) • Specific growth rates decrease, ribosomes degrade, central carbon metabolism altered -> affects RP production • How to avoid growth cessation, increase productivity, improve purification of RP

5. The system: cI857 mutant (1966): retains wild-type properties at low temperature, but unstable when temperature raised - Interactions of cI857 with operators released up to 37 C, > 37 C mutant repressor inactivated

6. History of pL/pR-cI857: • 1979:1st expression vectors using the pL promoter (production: 6.6% -> now 30%) • 1983: increased productivity through temperature-regulated runaway replication, plasmid with cI857 high compatibility • Other improvements: synthetic RBS, suitable poly-linkers, mutation to operator oR -> tight repression up to 39 C (Helicobacter) (2005) • Similar system in l. lactis using comparative molecular modeling of the known 3D structure of cI857

7. Molecular and Physiological responses after thermoinduction

8. Sigma 32 –master regulator • Sigma32 regulon includes almost all genes for proteins involved in folding and degradation (chaperones, proteases) • Temperature increase -> nucleotide misincorporation and chromosome damage; sigma32 activation -> DNA and RNA protected by members of the regulon; other regulon members transfer delta-3-isopentyl-PP to tRNA to stabilize codon-anticodon pairing to improve tRNA thermal resistance • overexpression and accumulation of unfolded recombinant proteins -> genes involved in protein folding and degradation respond; most of these controlled by sigma32

9. Post heat shock and recombinant protein accumulation • Initial rapid upregulation of genes for chaperons and proteases (some in minutes) -> unstable environment -> metabolic burden -> slow growth rate and quantity protein produced • High protein production -> a.a. depleted (min. media) -> deactylatedtRNAs bind to ribosome -> RelA recognizes and makes alarmones (p)ppGpp -> stringent response -> higher transcription of stress-related genes and translation process interrupted-> as above • Both limit RP production

10. Molecular and Physiological responses after thermoinduction

11. Transcriptomic analysis • Harcum and Haddadin: dual stress of heating above 37 C and accumulation of unfolded RP (heated 50oC and IPTG-induced) • Found: 163/1881 genes responded in dual stress vs. either heated or induced • Genes coding for RNA polymerase (eg. rpoA/S) and ribosome coding genes downregulated

12. Resulting physiological response after induction • Decrease in specific growth rate • Increase in respiration (RP production and hsp increase ATP requirements 6x) • Alteration of central carbon metabolism, glucose consumption

13. How to optimize heterologous protein production • Plasmid segregation • Host strain • Recombinant protein and localization • Culture strategies • Induction strategy – Heating duration and intensity

14. Plasmid segregation • Plasmid maintenance and replication -> metabolic load and consumption of resources (further drained upon induction of RP production) = plasmid-load • Plasmid-free cells favored at higher temperatures (derepressed). • In RP production: avoid plasmid segregation and extend the production phase after induction: maintain plasmid copy number with culture strategies

15. Culture strategies • Culture modes: batch, fed-batch and continuous • For plasmid copy# maintenance: • fed-batch (temporal): restrict specific growth rate to low values increasing rates of substrate addition before induction -> high cell concentrations • Continuous (spatial): higher plasmid stability and high cell density cultures in 1st , high RP productivity in 2nd (induced) • Lim and Jung: 23x final contration in fed-batch vs. batch culture (controlled substrate feed rate during growth phase and specific growth rate in production phase) • Curless et al.: 4-fold production under higher dilution rates tested – pre-induction specific growth rate affect productivity

16. Host Strain • Different e coli strains have different heterologous gene expression capacities • Protease-deficient: eg. BL21 most productive in a study • We use BL21s for expression

17. Recombinant Protein • Thermoinduced system’s response can lead to recombinant proteins being degraded • Comparison study suggests factors: RP’s proteolytic sensitivity and thermal lability

18. Protein accumulation and recovery Depending on localization signals: • Aggregates in the cytoplasm –IB easily isolated but have to refold after • Soluble form in cytoplsam • Soluble form in periplsamic – less proteolytic activity, simpler purification, fewer isoforms and post-trans. modifications, in vivo cleavage of signal peptide, formation of disulfide bonds • secreted to supernatant

19. Summary • Heat inducible system has many advantages but stresses cell out • Dual stress triggering of chaperone and protease production leads to comprised RP production • How to optimize productivity of RP

20. How different do you think internal cell responses are in other expression systems are? • How many of these possible stresses do we have to consider in our projects?