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Production of recombinant proteins in E. coli by the heat inducible expression system based on the phage lambda pJ and/or pR promotors

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

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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

why this paper
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
intro thermo regulated expression system for recombinant protein expression
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

cons focus of presentation
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
the system
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

history of pl pr ci857
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
sigma 32 master regulator
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
post heat shock and recombinant protein accumulation
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
transcriptomic analysis
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
resulting physiological response after induction
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
how to optimize heterologous protein production
How to optimize heterologous protein production
  • Plasmid segregation
  • Host strain
  • Recombinant protein and localization
  • Culture strategies
  • Induction strategy – Heating duration and intensity
plasmid segregation
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
culture strategies
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
host strain
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
recombinant protein
Recombinant Protein
  • Thermoinduced system’s response can lead to recombinant proteins being degraded
  • Comparison study suggests factors: RP’s proteolytic sensitivity and thermal lability
protein accumulation and recovery
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
summary
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
slide20

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?
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