Transcript Heat induced expression system ppt

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
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Chemical inducers (eg.
IPTG):
-expensive
-toxic
-Possible additional
controls to remove Systems based on
chemicals (esp . for nutrient exhaustion: (eg.
Depletion of an a.a.)
human use!)
- starvation affects cell
metabolism, synthesis of
the recombinant protein
- Precise control of
induction timing is
difficult
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?
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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
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
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
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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-isopentylPP 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
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-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
Harcum and Haddadin: dual stress of
heating above 37 C and accumulation
of unfolded RP (heated 50oC and IPTGinduced)
 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
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Decrease in specific growth rate
 Increase in respiration (RP production
and hsp increase ATP requirements 6x)
 Alteration of central carbon metabolism,
glucose consumption
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Plasmid segregation
 Host strain
 Recombinant protein and localization
 Culture strategies
 Induction strategy – Heating duration
and intensity
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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
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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
Different e coli strains have different
heterologous gene expression capacities
 Protease-deficient: eg. BL21 most
productive in a study
 We use BL21s for expression
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Thermoinduced system’s response can
lead to recombinant proteins being
degraded
 Comparison study suggests factors: RP’s
proteolytic sensitivity and thermal lability
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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
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
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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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