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 schoep@engineering.ucsb.edu, 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)

MKK ..HHHHHHHHHH.. Ala-X-Ala| - ..........C’Positively Charged

N-term"AXA Box"

HydrophobicRegion (10-20 aa)

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

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

ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11

Z= polar residue Φ = hydrophobic residues

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

ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11

cytoplasmic proteins

ER proteins

glycosylationdisulfide formatin

modification (eg .glycosylation)Packaging, distribution

chaperones

Recycle misfolded proteins

SUMMARY

As usual, prokaryotes are complicated enough!

ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11

Folded proteinsTAT pathway

Unfolded proteins Sec pathway

SRP pathway

Protein aggregationand degradation

Disulfide formation

Cytoplasmic proteins

oxidised

isomerase

Chaperone

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 – 11/10/11ChE 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 – 11/10/11ChE 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 – 11/10/11ChE 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 – 11/10/11ChE 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 – 11/10/11ChE 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 – 11/10/11ChE 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 chaperonesDna K- Dna J

Release form Dna K

Re-activate afterstress

Partially foldedproteins

Can form Inclusion bodies

ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11ChE 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 – 11/10/11ChE 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 – 11/10/11ChE 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 productionEg. Slowing of glycolytic flux to reduce acetate formation

Increase folding in periplasm

Co expression of chaperones and foldasesEg. 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 – 11/10/11ChE 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 genesEg. 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 cytoplasmEg. 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 – 11/10/11ChE 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

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

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!