2. Chapter 10 Highlights
Engineering Host Cells to manufacture proteins for mass
production
Increasing Efficiency
Transcription Systems
Translation
Activation Systems
mRNA expression and stability
Translational Control Systems
Codon Optimization
Protein Stability and Purification
Comparisons of Different Host Cell Expression Systems
Pre- and Post-Translational Modification Systems
Multiple Expression Systems
3. FIGURE 10.15
Comparison of Recombinant Protein Expression SystemsEach protein expression system falls
on a continuum of worst to best for characteristics such as speed, cost, glycosylation, folding,
and government regulations. Transgenic animals (rabbit) and transgenic plants (plants) are
discussed in Chapters 14 and 15The other symbols include mammalian cultured cells, insect
cell culture, yeast, and bacteria.
4. Recombinant Proteins
Proteins expressed from recombinant DNA gene
Detailed Study of Protein Expression
Reengineering of host DNA to produce desired proteins in mass
quantities
DNA Techniques
RNA Techniques
Protein Expression Techniques
Protein Purification and Production
Large Scale Protein Production
Clinically Relevant Proteins
Insulin, Interferon s, IL-2, Somatotropin, Erythropoietin, etc.
5. Recombinant Proteins
Pros
Pathway engineering is very specific for easy manipulation
depending on host cell and protein desired.
Greater copy number of genes results in higher quantity of
product
Can use high-copy plasmids
Prevent plasmid loss by genome integration of DNA
Cons
Large scale production and purification is extremely difficult and
precise
High-copy plasmids may be unstable or redundancy may occur
Can be difficult to integrate multiple copies of gene into host
genome due to unreliability of multiple gene copy integration
6. Recombinant Protein Process
Determining DNA, RNA, Protein sequences
Cloning of correct gene into Expression Vector for enhanced
production
Sequencing techniques
PCR and RT-PCR
gDNA and cDNA Libraries
Restriction Endonuclease Digestions
Gene Intregation and Ligation into Vector
Transformation of Vector into Host Cell and Expression
Cold Shock and Ca Treament for Transformation
Gene Intregation into gDNA
Heat Shock
8. Prokaryotic vs Eukaryotic Cell Use in
Protein Expression
Prokaryotic Cells
Easiest cells to grow and genetically manipulate
Antibiotic resistance genes for increased selectivity of transformed
bacteria
Lack before and after-translation protein modification pathways for
correct protein manufacturing
Eukaryotic Cells
Not all genes are able to be expressed in prokaryotic cells
Has all necessary promoters and terminators in gDNA already
9. mRNA Factors
Strength between mRNA Ribosome Binding Site and Ribosome
interaction
mRNA Stability and Structure
Codon usage
Prevention of mRNA secondary structure overlap or folding
Correct formation of poly A tail and methyl-G cap
10. FIGURE 10.15
Comparison of Recombinant Protein Expression SystemsEach protein expression system falls
on a continuum of worst to best for characteristics such as speed, cost, glycosylation, folding,
and government regulations. Transgenic animals (rabbit) and transgenic plants (plants) are
discussed in Chapters 14 and 15The other symbols include mammalian cultured cells, insect
cell culture, yeast, and bacteria.
11. Translation Expression Vectors
Vector provides the most optimal
ribosomal binding site
Strong consensus RBS and 8 bp
space between RBS and Start
codon for increased binding
affinity and improved translation
mRNA may back onto RBS region
depending on sequence
12. Codon Usage Rate
Engineering of DNA sequence for codon optimization
Alter DNA sequence for improved translation effeciency
Can limit translation if tRNA anticodons used for amino acids are
not in abundance
Ex. Lysine encoded by AAA 25% and AAG 75%. Figure 10.3 has E.
coli waiting on UUU tRNA since it mainly uses AAG as primary
codon.
Directly supply rare tRNA for increased translation
Can be very expensive depending on scale of production
13. FIGURE 10.3
Codon Usage Affects Rate of TranslationBacteria prefer one codon for a particular amino acid to other
13
redundant codons. In this example, the ribosome is stalled because it is waiting for lysine tRNA with a
UUU anticodon. Escherichia coli does not use this codon very often and there is a limited supply of
this tRNA.
14. Toxic Effects of Protein
Overproduction
Overproduction of proteins may condense into an aggregate of
misfolded and nonfunctional proteins called Inclusion Bodies
Inclusion bodies result in a decrease in efficiency and waste of
resources
Results from limitation in protein processing and natural timedependent degradation of proteins.
15. pET Vector Expression System
Use of vector expression system for protein production control to
increase efficiency and mitigate inclusion bodies
pET Vector Expression System consists of 4 Sites:
Normal Function – No Protein Expresion
Site of transcription with lac operon and gene of interest
Origin of Replication and Antibiotic Resistance Gene
Lac I for production of Lac operon repressor protein
Lac I protein represses transcription by preventing T7 RNA Polymerase
expression
Altered Function – Protein Expression
IPTG is added to induce protein expression
IPTG binds to Lac repressor protein and expresses T7 RNA Polymerase for
transcription
16.
17. pBAD Expression System
Expression system based
on Arabinose Operon
Normal Function – OFF
AraC regulatory proteins
bind O2 and O1 sites and
create dimer
Addition of Arabinose –
ON
AraC binds to I site and
activates transcription
Transcription increase is
dose-dependent
18. Protein Stability
Factors in Protein Stability and Degradation
Natural Degradation or time left unprocessed
Overall 3D Structure
N-end Rule
Prokaryotes – Val, Met, Ala, etc – 20 hr, and Arg – 2 min
Humans – Val – 100 hr, Met/Gly – 30 hr, and Glu, Arg – 1 hr
Easy to alter through DNA Sequence to produce longer lasting free proteins
Pest Sequences
Regions rich in (P) Proline, (E) Glutamine, (S) Serine, and (T) Threonine
Very recognizable by proteosomes
Most difficult to alter these sequences due to internal sequence change
that can disrupt final protein function or disrupting protein synthes
Alter final protein function or make protein nonfunctional
Disruption of protein synthesis or make protein unstable during synthesis
19. Protein Stability
Addition of Moleculer Chaperones to mitigate formation of inclusion
bodies
Molecular chaperones bind free amino acids of the growing
polypeptide chain before folding
20. Improving Protein Secretion
Protein Synthesis can terminate anywhere in the cell
Protein Secretion can be engineered to arrange for optimal destination
Periplasmic Space
2 - Type 1 Secretory System
Use of Transmembrane proteins that are active/passive transporters
Hydrophobic signal at the N-terminal
3 Types of Secretory Systems:
1 - General Secretory System
Cytoplasm, plasma membrane, extracellular matrix
Transmembrane domain to outside of cell
3 – Type 2 Secretory System
Periplasmic Space and then outer membrane transport to outside of cell
21. General Secretory System
Transports protein into periplasmic space
Allows protein extraction harvest from cell
Aggregate of Inclusion bodies may occur if there is overproduction of
protein
Increase of secretory proteins into inner membrane can be used to
decrease inclusion bodies
22. Type 1 Secretory System
Transport of protein through periplasmic space to the outside of the
cell by a transmembrane protein that spans entire membrane
Protein may have hydrophobic signal sequence at N-terminal for
simple transport
Protein Fusion may be used to transport across
Fusion of Normal protein and Bacterial protein that can be transported
across membrane
Binding maltose protein to normal protein for transmembrane delivery
Cleave maltose after transport by proteases
23. Type 2 Secretory System
Two Step System
Transport of protein into periplasmic space by general secretory
system
Transport of protein from periplasmic space to the outside of cell by an
outermembrane protein
Combination of general and Type 1 systems
Specific export of protein outside of cell
24.
25. Protein Fusion Expression
Vectors
Plasmid that links or binds TWO proteins together for various
purposes.
Example: MalE Protein
Assemblage at N-terminal or C-terminal
Mainly for secretion, but also Solubility, Stability
Protein fused to MalE within cell
Transport of fusion protein to Periplasmic space by maltose induction
Pre-made Fusion Expression Vector
Mix and Match Fusion Proteins through pBAD expression control
26. Protein Fusion Expression
Vector Examples
Simple Protein Fusion Vector
Single Vector with attachment
to thioredoxin protein
CM4 is GoI
ProAsp gene is for peptide
cleavage site
His-tag is for purification
http://www.springerimages.com/Images/LifeSciences/1-10.1007_s10529-007-9351-4-0
28. Eukaryotic Cell Expression
Post-translational modification systems in Eukaryotes
Novel Amino Acids in protein sequence
Glycosylation for cell surface recognition and function retention
Addition of other chemical groups
Fatty Acid Chains – lipids
Acetyl Groups Phosphate Groups – DNA , RNA, phosphorylation
Disulfide Bonds
Cleavage sites
30. Yeast Protein Expression
Pros
Cons
Similar to bacterial protein expression
Naturally occuring plasmid
Secretes few proteins for easy purification of recombinant protein
Able to carry out many post-translational modifications
Loss of expression plasmids in large bioreactors
Only glycosylates secreted proteins (can be altered)
Addition of signal sequence to recombinant protein for secretion and
purification (Fig 10.9)
Similar to protein fusion
32. Expression of Proteins in
Insect Cells
Insect cells are simple and cheap to grow with many of the added
benefits of using mammalian cells
Vectors are Baculoviruses
Baculovirus infects insect cells and take control of cell for viral protein
production
After host death, baculovirus embeds viral particles in protein matrix
(capsule) called Polyhedrons
Polyhedrin is not needed. Transfer gene of interest to Baculovirus at this
site
Main baculovirus is the Multiple Nuclear Polyhedrosis Virus (MNPV)
Broad spectrum baculovirus
High yield of polyhedrins
34. Bacmid Shuttle Vector
Baculovirus expression vectors may give undesirable results
Bacmids created as a shuttle vector for alternate use of infecting insect
cells
Baculovirus-plasmid hybrid
Contains E. Coli origin, cloning site, and antibiotic resistance site
Allow bacmids to survive in E. coli and infect insect cells
Figure
10.11
35. Insect Cell Expression
Disadvantage
Glycosylation pathway is different in Insect Cell lines in mamalian cell
lines
Insect Cells – Mannose derivative pathway
Mammalian Cells – Full glycosylation pathway of sialic acid derivatives
Fig. 10.12
36. Expression of Proteins in
Mammalian Cells
Most complex method of engineering for mammalian cells
Mammalian Shuttle Vectors include:
Bacterial origin of replication and antibiotic resistance
Selection at prokaryotic level
Strong viral or mammalian promoters
Multiple cloning sites
Types of selective genes for mammalian cell growth
Antibiotic Selective Gene
Enzymatic Selective Gene
Geneticin – blocks protein synthesis
Npt gene inactivates antibiotic
k
DHFR Gene knockout host cells
37. Mammalian Cell Selection
Three Types of selective genes for mammalian cell growth
Antibiotic Selective Gene
Geneticin – blocks protein synthesis
Npt gene inactivates antibiotic
Enzymatic Selective Gene
Host Cell knockout of DHFR gene
DHFR – cofactor of folic acid and inhibited by methotrexate
DHFR gene is included on plasmid
Methotrexate inhibition for high-level expression selection
Metabolic Selective Gene
Glutamine synthetase enzyme is included in shuttle vector
Select cell lines by addition of methionine sulvoximine
Mammalian cell selection with multicopy plasmids
39. Expression of Proteins with Multiple
Subunits in Mammalian cells
Expression of proteins with multiple subunits can be difficult to
produce because assembly of protein outside of cell is very difficult
Three Methods for multiple subunit expression:
Multiple vectors are used with a single gene copy of each subunit
1.
2.
Co-expression of genes on a single vector with two separate
promoters
3.
Assembly must occur outside of cell
Creates two monocistronic mRNA’s
Co-expression of genes on a single vector with a single promoter
and an IRES between genes
Creates one polycistronic mRNA
Two ribosomes read the same mRNA for multiple subunit translation
41. FIGURE 10.15
Comparison of Recombinant Protein Expression SystemsEach protein expression system falls
on a continuum of worst to best for characteristics such as speed, cost, glycosylation, folding,
and government regulations. Transgenic animals (rabbit) and transgenic plants (plants) are
discussed in Chapters 14 and 15The other symbols include mammalian cultured cells, insect
cell culture, yeast, and bacteria.