2.  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. Heat- inducible expression system pros: - λ pL/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? Chemical inducers (eg. IPTG): -expensive -toxic -Possible additional controls to remove chemicals (esp. for human use!) 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.  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. 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.  1979:1 st 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

8.  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. -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) -> deactylated tRNAs 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

11.  Harcum and Haddadin: dual stress of heating above 37 C and accumulation of unfolded RP (heated 50 o C 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.  Decrease in specific growth rate  Increase in respiration (RP production and hsp increase ATP requirements 6x)  Alteration of central carbon metabolism, glucose consumption

13.  Plasmid segregation  Host strain  Recombinant protein and localization  Culture strategies  Induction strategy – Heating duration and intensity

14.  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 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 1 st, high RP productivity in 2 nd (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.  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.  Thermoinduced system’s response can lead to recombinant proteins being degraded  Comparison study suggests factors: RP’s proteolytic sensitivity and thermal lability

18. 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.  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?