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ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
The Cell as a Production Factory, Expression Systems
Tobias Schoep
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
No chapter reference. Biology background, Chapter 15
Questions to [email protected], Rm 3114 or contact Serra Elliot
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Cells as production factories
Cellular method of protein production depends on protein properties
Different systems have different advantages and disadvantages for protein production
To understand the advantages and disadvantages of expression systems we must
understand a bit more biology
Biology of protein folding, transport and modification – a brief overview
Prokaryotic protein expression systems
Eukaryotic protein expression systems
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Protein Production
Prokaryotes: Translation in the cytoplasm
Eukaryotes: Translation in the cytoplasm and endoplasmic reticulum
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Protein Folding (Eukaryotes)
Proteins are produced in cytosol and endoplasmic reticulum
Endoplasmic reticulum targeting requires a signal sequence
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Protein Folding (Eukaryotes)
Endoplasmic reticulm
Function: Protein folding, glycosylation and export to golgi apparatus (eukaryotes)
Correct folding eg. disulfide bond formation mediated by chaperones
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Protein Transport (Eukaryotes)
Proteins move from ER to golgi apparatus in vesicles
Golgi apparatus function: protein modification, packaging for distribution
misfolded proteins
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Protein Transport (Eukaryotes)
Lectures to come:
Trafficking of proteins through cells
Transport through membranes: facilitated and passive
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Protein Modification (Eukaryotes)
Glycosylation
Formation of protein / carbohydrate complex
Important for protein structure, function and targeting
Aberrant glycosylation can cause disease
eg. Congenital Disorders of Glycosylation
Many proteins glycosylated in ER
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Protein Modification (Eukaryotes)
Glycosylation
Additional modifications to glycoproteins in
golgi apparatus
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Protein Folding (Prokaryotes)
Proteins are produced in cytoplasm
Cytoplasm is a mildly reducing environment
Disulfide bond formation in periplasm
Protein folding in cytoplasm can be mediated by chaperones
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Protein Transport (Prokaryotes)
Protein transport is more complex in eukaryotes
Transport is also important in prokaryotes eg. secreted proteins
Targeting proteins to periplasm for disulfide bond formation
Periplasmic targeting requires a signal sequence (signal peptide)
Hydrophobic
Region (10-20 aa)
MKK ..HHHHHHHHHH.. Ala-X-Ala| - ..........C’
Positively Charged
N-term
"AXA Box"
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Protein Transport (Prokaryotes)
There are at least 6 secretion systems
Specific secretion systems are often bacterial species specific
The type II secretion system (T2SS) is main secretion apparatus
Sec /TAT /SRP
complex
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Protein Transport (Prokaryotes)
In E.coli there are 3 types of T2SS from the cytoplasm to the periplasm
Sec pathway: transport of unfolded proteins
TAT pathway: transport of folded proteins
SRP pathway: protein translated directly into periplasm
Different signal sequences target proteins to secretion pathways
Z= polar residue
Φ = hydrophobic residues
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Protein Modification (Prokaryotes)
Most bacteria do not glycosylate
If glycosylation is important in protein function, bacterially produced protein will not
function
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
SUMMARY
As usual, eukaryotes are more complicated
cytoplasmic proteins
Recycle misfolded proteins
ER proteins
glycosylation
disulfide formatin
modification (eg .glycosylation)
Packaging, distribution
chaperones
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
SUMMARY
As usual, prokaryotes are complicated enough!
Protein aggregation
and degradation
isomerase
Disulfide formation
oxidised
Folded proteins
TAT pathway
Cytoplasmic proteins
SRP pathway
Chaperone
Unfolded proteins Sec pathway
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Biology of protein production, folding and modification
Prokaryotic protein expression systems
Eukaryotic protein expression systems
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Prokaryotic protein expression systems
Escherichia coli (E.coli)
Used for production of first recombinant DNA biopharmaceutical (Insulin) by Eli Lilly
Production of bovine growth hormone (bGH) on ton scale by Monsanto in 1994 ($11.60/g)
(both Insulin and bGH require oxidative folding)
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Advantages of E.coli for protein production
Rapid growth, on inexpensive carbon source
Amenable to high density fermentation and scale-up
Genetics very well characterized, chromosome sequenced
Many tools for genetic manipulation/ cellular engineering
Disadvantages of E.coli for protein production
Cytoplasm is a mildly reducing environment
Does not perform post-translational modifications eg. glycosylation
Does not allow folding of complex proteins with multiple disufides
Complex proteins often form inclusion bodies
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
What happens when proteins fold partially?
Aggregate and may form inclusion bodies
Degrade
Eventually fold correctly
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Considerations when producing proteins in E.coli
Gene and codon usage
Transcriptional & Translation Regulation
Protein Folding and Targeting
Host Engineering: Chaperones & Proteases
Culture Conditions
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Gene and codon usage
Organisms show preference for codon (mRNA) that codes an amino acid
Design genes to have optimal E. coli codon usage
Codon usage reflects tRNA pool available for translation
Produce codon optimized gene synthetically
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Transcriptional & Translation Regulation
Low gene dosage/copy number
Tightly-regulated promoters eg. arabinose operon
Enhanced mRNA stability
Optimized translational initiation eg. optmized Shine Dalgarno sequence
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Protein Folding and Targeting
Cytoplasm is a mildly reducing environment
Disulfide bond formation mainly in periplasm
Protein folding in cytoplasm can be mediated by chaperones
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Protein Folding and Targeting
Protein folding in cytoplasm can be mediated by chaperones
Dna K- Dna J
Can form
Inclusion bodies
Re-activate after
stress
Partially folded
proteins
Release form Dna K
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Protein Folding and Targeting
Does the protein need to be secreted or expressed in the cytoplasm
Cytoplamic expression- batch cultures
Secreted proteins- continuous cultures
Selection of appropriate secretion system considering disulfide formation requirements
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Protein Folding and Targeting
Problems with secreted proteins:
Incomplete processing of signal peptides
Variable secretion efficiency
Slow rate of accumulation (degradation?)
Formation inclusion bodies
Incorrect disulphide formation
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Host Engineering: Chaperones & Proteases
Alter metabolism to favor protein production
Metabolic engineering of pathways influencing protein production
Eg. Slowing of glycolytic flux to reduce acetate formation
Increase folding in periplasm
Co expression of chaperones and foldases
Eg. Seventeen Kd Protein (Skp) chaperone assists OMP folding, increased
functional yield of scFv fragments in periplasm
Eg. Protein Disulphide Isomerase
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Host Engineering: Chaperones & Proteases
Reduce proteolytic degradation of proteins
Knockout of protease genes
Eg. Cytoplasmic Proteaes: Lon, Clp (A,X,Y,P, YQ)
Eg. Periplasmic proteaes: DegP, Prc (Tsp)
Eg. Membrane proteases: DegS, DegQ, Protease III,
Increase folding in cytoplasm
Engineering strains with altered redox environment in cytoplasm
Eg. ORIGAMI (Novagen) allowed production of Human Tissue Plasminogen Activator with 17
dsbs.
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Culture Conditions
Often protein specific
Optimize substrate feed, temperature, induction conditions
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Biology of protein production, folding and modification
Prokaryotic protein expression systems
Eukaryotic protein expression systems
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Eukaryotic systems for protein production
Yeast - Saccharomyces cerevisiae, Pichia pastoris
Insect Cells (e.g., SF9)
Mammalian Cells (CHO, NS0, MS2, Hybridoma)
Plants
Transgenic Animals
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Considerations when producing proteins in eukaryotic systems
Gene and codon usage – NO
Transcriptional & Translation Regulation – YES – Tet ON, Tet OFF systems
Protein Folding and Targeting – NO
Host Engineering – YES
Culture Conditions – YES
Improved environmental control i.e., Temp, pH, fed-batch, media additives
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Yeast
Unicellular organism
As a eukaryote share the complex internal cell structure of plants and animals
Can be grown in liquid culture like bacteria
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Advantages of Yeast (S.cerevisiae) for protein production
Rapid growth, on inexpensive carbon source
Amenable to high density fermentation and scale-up
Genetics very well characterized, chromosome sequenced
Many tools for genetic manipulation/ cellular engineering
Can form correct disulfide bonds for eukaryotic proteins
Can secrete proteins
Disadvantages of Yeast (S. cerevisiae)
Only simple glycosylation and hyperglycosylation (large mannose glycans are
problematic for human therapeutics)
Sometimes low expression levels (<mg/L)
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Host Engineering
GlycoFi
Introduce human glycosylation pathways in to Yeast (P. pastoris)
Localized synthetic enzymes fusions in ER
Produced complex human glycoproteins in yeast
Technology purchased by Merk for $400M in 2006
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Mammalian cells
Derived from a mammal
Immortalized cells, such as cancer cells
Generally adapted to suspension culture for protein production
Common cell lines for protein production
CHO – Chinese hamster ovary cells
293 - Human kidney 293 cells
Hybridoma cells for antibody production (previous lecture 10/18/11)
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Advantages of mammalian cells for protein production
Correct glycosylation of proteins (although can between cell types)
Correct disulfide bridges formed
Disadvantages of mammalian cells
Slow growth rates
Production of stable engineered cells takes up to 6 months
Low yields (mg/L)
Upscaling protein production can be problematic
Some concerns with safety as many cell lines are cancer derived and viral vectors used
for engineering
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Timeline for mammalian cells for protein production
See lecture Manipulating genes, cellular engineering
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Host Engineering
Metabolic engineering of pathways influencing protein production
E.g. Reduction of lactate production. Lactic acid can inhibit cell growth and affect cellular
metabolism at high concentrations.
Metabolic engineering of pathways influencing cell survival
E.g. Prevention of apoptosis (programmed cell death) by overexpression of Bcl-2. Inhibits
formation of Mitochondrial Outer Membrane Permeabilization Pore.
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
CHO cells used for biologicals
ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11
Good luck in the quiz!