For expression of gene in a particular vector, always used strong regulatable promoter (lac promoter, trp promoter, tac promoter , trc promoter, pL promoter, T7 gene promoter) use of dual plasmid system & fusion proteins How we can increase our protein product yield?

1. PROKARYOTIC EXPRESSION
SYSTEMS
Selected Reading:
Chapter 6
Manipulation of gene expression in prokaryotes
Glick B. R. et al. 2010. MOLECULAR BIOTECHNOLOGY (4th Edition)

2. Background knowledge required:
• Transcription
• Operon
• Vectors
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3. Expression vectors
Ori RBS TT
The molecular biological features that have been manipulated to modulate gene expression
include;
• the promoter and transcription terminator sequences,
• the strength of the ribosome-binding site,
• the number of copies of the cloned gene and whether the gene is plasmid borne or integrated
into the genome of the host cell,
• the final cellular location of the synthesized foreign protein,
• the efficiency of translation in the host organism, and
• the intrinsic stability within the host cell of the protein encoded by the cloned gene.
There is no single strategy for obtaining maximal expression of every cloned
gene. Consideration of the distinctive features of a cloned sequence is usually
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4. Potential Problems in Prokaryotic Expression Systems
• Low expression
• Degradation by bacterial proteases
• Improper folding
• Oxygen limitations
• Biofilm formation
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5. Strong Promoters
• Strong promoters
• High affinity for RNA polymerase, for frequent transcription of its gene
• Constitutive promoters
• Continuously expressed
• Drawbacks:
1. Energy drainage
2. Plasmid instability
It is important to control transcription for cloned gene expression at SPECIFIC STAGE
and SPECIFIC DURATION
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6. Strong Regulatable Promoters
• Molecular switches
1. lac promoter
2. trp promoter
3. tac promoter
4. trc promoter
5. pL promoter
6. T7 gene 10 promoter
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7. 1. lac promoter
• Inducer
• Lactose Allolactose (β-galactosidase)
• IPTG (Isopropyl thiogalactopyranoside)
Lactose Lac promoter Repressor protein
binding
- OFF +
+ ON -
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9. Transcription regulation by lac promoter
• There is an increase in gene expression after;
• Binding of CAP (Catabolic Activator Protein) to CAP box
• Binding of cAMP to CAP
• Example: lacUV5
• Altered nucleotide sequence at -10
• Stronger than wild type lac promoter
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11. 2. trp promoter
• Negatively regulated
• Inducers
• Trptophan
• 3-indoleacrylic acid
• Drawback:
• Leaky
• Not suitable for potential toxic/deleterious genes
Trptophan Lac promoter Repressor protein
binding
- ON -
+ OFF +
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12. 3. tac and trc promoters
• Hybrid construct
• Spacer region between -10 and -35 is
• 16 bp for tac
• 17 bp for trc
• 3x stronger than trp
• 10x effective than lac
tac
trp lac
-35 -10
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13. Importance of spacer region
• Contributes to the strength of promoter
• -13 to -20 of lac promoter
• Replacement of GC rich region with AT rich
• pR 2x stronger expression
• Altered promoters
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14. 4. pL promoter
• Temperature regulated
• Repressor protein:
• cI857, thermosensitive
Temperature Repressor
protein
Pl promoter
28ᵒC ACTIVE OFF
42ᵒC INACTIVE ON
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15. 5. T7 gene 10 promoter
• Requires integration of T7 RNA polymerase gene integration into E.
coli chromosome under control of lac promoter
• Lag of 1 hr after addition of inducer
• Examples:
• pET vectors
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16. Effectiveness of deactivating repressor
protein
• Depends on
• Ratio of number of repressor protein to number of promoter sequence
• For complete control
• Two plasmids
1. Repressor protein
2. Promoter of cloned gene
• Example: lacIq
• Mutant
• controls leakiness by producing high levels of lac repressor
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17. How to increase cloned protein production
• pCP3
• Created to obtain highest
possible level of foreign
protein
• Increasing copy number
5-10 fold (42 ᵒC)
• By replacing its DNA
replication origin with that
from plasmid pKN402
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20. Large scale protein production systems
• Bioreactors
• Small vessels
• Pilot plant size
• Industrial scale
• Problems:
• Thermal/chemical induction
• Time consuming
• Costly
• Solution:
• Two/Dual plasmid system
• Chemical inducers;
• Costly
• Toxic
• Difficult to remove
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21. With no tryptophan in the medium
With tryptophan in the medium
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22. Dual plasmid system
• Advantages:
• Can be grown on inexpensive medium
• Molasses and casein hydrolysate
• Example:
• Β-galactosidase 21%
• Citrate synthase 24%
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23. Choice of promoter for large scale production
• Considerations;
• Thermal induction may induce hsp
• Nutrient promoter limits choice of media
• O2 promoters need a specific dissolved oxygen level
• House keeping δD vs. stationery phase δS
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24. • The results indicated
1. All the promoters are active to some extent in each of the bacteria tested
2. In E.coli, tac is the most active promoter
3. Nm is the most active promoter in all other bacteria
Expression in other microorganisms
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25. Example: Lactic acid bacteria
• Lactococcus spp.
• Any change must not affect
• The production process
• Product palatability
• Appearance
• Construction of plasmid library (synthetic plasmids)
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26. FUSION PROTEINS
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27. Fusion proteins
• Problem:
• Foreign proteins are prone to degradation……low
expression
• Solution: fusion proteins
• Engineer a contruct that encodes for the target
protein in frame with stable host protein
• Fusion protein protects the cloned gene product
from attack by host proteases
• Construct should maintain the correct reading
frame
• Table 6.2
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29. Uses of fusion proteins
• For reducing degradation
• For increased and simplified purification
• Examples:
1) IL-2
• Marker peptide sequence
• N-D-Y-K-D-D-D-D-K-C
• Fig 6.12, 6.13
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30. • Fig 6.15
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32. Use of fusion proteins
2) Use of His-tag
• 6-8 his residues on either N- or C- terminal
• Passed over an affinity column of nickel-nitrilotriacetic acid
• Elution by imidazole
• 100x production
• 90% recovery
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33. Cleavage of fusion proteins
• Proteases used;
• Enterokinase
• Tobacco etch virus protease
• Thrombin
• Factor Xa
• Intein
• Internal segment of a protein that, under specific conditions, catalyzes its own
cleavage into two separate polypeptides
• 100 known inteins
Cys/Ser AspN C
• Oligonucleotide linkers encoding
N-Ile-Glu-Gly-Arg-cloned protein-C
• Fig 6.14
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34. • Purification of a protein of interest from an intein-containing fusion
protein bound to a chitin chromatography column through a chitin-
binding domain.
• Cleavages occurs upon the addition of dithiothreitol
Used for:
1. Cre recombinase
2. α-1-antitrypsin
3. Human fibroblast
growth factor
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37. Surface display
• Specialized fusion protein system…….for cDNA libraries
• cDNA cloned into surface protein gene of filamentious
bacteriophage/bacterium
• Immunological assay can identify
• Fusions with PIII of M13 bacteriophage
Surface protein gene
(filament or pilus protein)
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N-M13 packaging DNA-lac promoter-pIII gene-GOI-C

38. Surface display on bacteria
• Fusions between the genes for the
target protein and for an outer surface
protein are created to export proteins
to the surface of a gram-negative
bacteria
• Bacterial fusion partners include:
• OmpA….. E. coli
• PAL……E. coli
• OprF…..Pseudomonas aeruginosa
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Surface displayed fusion proteins as vaccines
e.g. epitope of P. falciparum (malarial parasite) Asn-Ala-Asn-
Pro inserted on OprF’s surface exposed loops worked reacted
positively

39. TRANSLATION EXPRESSION
VECTORS
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40. Translational expression vectors
• Problem: putting the cloned gene under a strong, regulatable
promoter, although essential, may not be sufficient to maximize the
yield of cloned gene.
• Other factors;
• Efficiency of translation
• Stability of cloned gene product
• Solution: Translation expression vectors
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41. Translational expression vectors
• RBS (ribosome binding site)
• Sequence of 6-8 nts that can base pair with RNA of small
ribosomal subunit
• 5’-UAAGGAGG-3’
• Strong binding of mRNA to ribosomal subunit…..greater
translational initiation efficiency
• Other considerations;
1. rbs….at precise distance to start codon
2. No localized secondary structure
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42. Translational expression vectors
• pKK233-2
• Ampr gene….selectable marker
• Tac promoter
• Rbs from Lac Z
• ATG start codon….8 nt downstream of rbs
• Transcription terminators T1 & T2…..λ
• MCS includes NcoI, PstI & HindIII
No single optimized translation initiation region
• N-terminal region vary from protein to protein
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43. Rarely used codon
• Least used codons:
• AGG……Arg
• AGA…..Arg
• AUA….Ile
• CUA…..Leu
• CGA…..Arg
• Problem: Presence of 2 or more rarely used codons close to each other or
adjacent or in N-terminal is detrimental to protein expression
• Solution:
1. If the target gene is eukaryotic, it may be cloned and expressed in a eukaryotic
system
2. Codon optimization
3. A host cell that has been engineered to overexpress several rare tRNA may be
employed (Fig 6.20)
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44. • Commercially available
E. coli strain for the
overproduction of tRNA
• argU…..AGG/AGA
• ileY……AUA
• leuW……CUA
• 100x of Ara h2 (peanut
allergen)
• Table 6.3
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45. • Table 6.3
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46. INCREASING PROTEIN STABILITY
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47. Intrinsic protein stability
• Half life of different proteins vary….differential stability
• Extent of disulphide bond formation…..increases stability
• Presence of certain aa at N- terminus….. increases stability
• Presence of internal aa (PEST sequence)….. decreases stability
• Table 6.4
• PEST sequence
• Rich in proline (P), Glutamic Acid (E), Serine (S) and Threonine (T)
• Often flanked by positively charged aa, Lysine (K) and Arginine (R)
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49. Facilitating protein folding
• Problem: Proteins produced in E. coli
accumulate in the form of insoluble,
intracellular, biologically inactive inclusion
bodies because of incorrect folding
• The extraction procedure requires
expensive and time consuming protein
solubilization and refolding procedures
• Solution: Fusion proteins that contain
thioredoxin as the fusion partner remain
soluble
In absence of trptophan
In presence of trptophan
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50. Facilitating protein folding-II
• Problem: Foreign proteins that contain 3 or
more disulfides generally do not fold
correctly in bacteria and often form
inclusion bodies
• Solution 1: The gene encoding human
tissue plasminogen activator (tPA) was
coexpressed with gene for either rat or
yeast protein disulfide isomerase to assist
protein folding
• Solution 2: Overproduction of (Disulfide
bond forming protein) DsbC results in
correctly folded and active human tPA
• Solution 3: Overproduction of all four Dsb
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51. Coexpression strategies
•The expression of foreign proteins in E. coli results in the formation of
inclusion bodies of inactive proteins at 37 ºC
•Cultivation of recombinant strains at low temperatures, resulting in
proper protein folding, often significantly increases the amount of
active protein
• However, E. coli (mesophile) grow very slow at low temperatures
•Recombinant strain of E. coli containing the chaperonin 60 gene and
cochaperonin 10 gene can grow at 4 to10ºC
•However, this is the first step of expressing system for temperature
sensitive proteins
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52. OVERCOMING OXYGEN
LIMITATION
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53. Protease-Deficient Host Strain
•One possible way to stabilize foreign proteins produced in E. coli is to
use host strains deficient of proteolytic enzymes
•However, this is not as simple because E. coli has at least 25 different
proteases, and only a few have been studied
•These enzymes are necessary for the degradation of abnormal or
defective proteins
•Thus, decreasing protease activity caused cells to be debilitated
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54. Bacterial Heamoglobin
•Some strains of Vitreoscilla bacterium normally live in oxygen-poor
environments
•These bacteria synthesized a hemoglobin-like molecule that binds
oxygen from environment and increases the level of available oxygen
inside the cells
•When the gene was cloned and expressed in E. coli, the transformants
displayed higher levels of synthesis of both cellular and recombinant
proteins, higher level of cellular respiration, and higher level of ATP
contents
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57. LIMITING BIOFILM FORMATION
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58. Biofilms
•Problem: The bacterial cells typically attach
to a surface, form a monolayer, and later
organize into a biofilm, a mixture of
bacterial cells and polysaccharides
•These cells are difficult to transform with
plasmid DNA, and are typically resistant to
high levels of antibiotics
•The foreign protein production is limited
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59. • Solution: Recombinant E. coli with deleted genes of biofilm
synthesis
• Pili….for initial attachment of bacteria cell to solid surface
• curli….for cell-cell and cell-surface attachment
• colanic acid….for three dimensional structure of biofilm
• Transformed bacteria are sensitive to antibiotics and produce
a higher level of recombinant protein.
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60. DNA INTEGRATION INTO HOST
CHROMOSOME
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61. Problem:
•High-copy-number plasmids impose a greater metabolic load than do
low-copy-number plasmids.
•A fraction of the cell population often loses its plasmids during cell
growth, diminishing the yield of cloned gene product .
•On a laboratory scale, plasmid-containing cells are maintained by
growing the cells in the presence of either an antibiotic or an
essential metabolite that allow only plasmid-containing cells to thrive.
•However, it is costly and difficult in the large-scale production.
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62. Solution:
•The introduction of cloned DNA directly into chromosomal DNA of
the host organism can overcome the problem.
•When DNA is part of the host chromosomal DNA, it is relatively stable
and consequently can be maintained for many generations in the
absence of selective agents.
•Important concerns;
1. The chromosomal integration site of a cloned gene must not be within an
essential coding gene.
2. The input DNA sequence must be targeted to a specific nonessential site
within the chromosome.
3. The input gene should be under the control of a regulatable promoter.
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63. Protocol for DNA integration
• DNA sequence similarity (~50 nt)
1. Identify the desired chromosomal
integration site
2. Isolate and clone part or all of the
chromosomal integration site
3. Ligate a cloned gene and a
regulatable promoter either into or
adjacent to the chromosomal
integration site
4. Transfer the construct into the host
cell as part of a non replicating
plasmid
5. Select and perpetuate the host cell
expressing cloned protein
into
adjacent to
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64. • The researchers constructed an E. coli plasmid that
contained an α-amylase gene from Bacillus
amyloliquifaciens that had been inserted into the middle
of chromosomal DNA fragment from B. subtilis but could
not replicate in B. subtilis.
• B. subtilis transformants expressing α-amylase are
selected.
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66. Removal of selectable marker gene
•The presence of selectable marker gene for antibiotic resistance in a
genetically modified organism that is released into the environment is
not desirable.
•The Cre-loxP recombination system, consists of the Cre recombinase
enzyme and two 34-bp loxP recombination sites, is employed.
•The marker gene to be removed is flanked by loxP sites, and after
integration of the plasmid into the chromosomal DNA, the marker is
removed by the Cre enzyme.
•The Cre enzyme is under of the control of lac promoter and can be
removed by shifting the temperature
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67. Cre-loxP recombination system
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68. INCREASING SECRETION
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69. Secretion into periplasm
•Directing a foreign protein to the periplasm or the growth medium
makes its purification easier and less costly, as many fewer proteins
are present there than in the cytoplasm.
•Recombinant proinsulin is approximately 10 times more stable if it is
secreted (exported) into the periplasm.
•Secretion of proteins to periplasm facilitates the correct formation of
disulfide bonds because the periplasm provides an oxidative
environment, in contrast to the more reducing environment of the
cytoplasm
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71. • The signal peptide at the N-terminal end facilitates its export
by enabling the protein to pass through the cell membrane.
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72. Increasing Secretion
Secretion into the Periplasm
• However, the presence of a signal peptide sequence
does not necessarily guarantee a high rate of secretion.
• The interleukin-2 gene downstream from the gene for
the entire propeptide maltose-binding protein, rather
than just the signal peptide, with DNA encoding the
factor Xa recognition site as a linker peptide separating
the two genes.
• As expected, a large fraction of the fusion protein was
found to be localized in the host cell periplasm.
• Functional interleukin-2 could then be released by
digestion with factor Xa.
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75. Increasing Secretion
Secretion into the Medium
• E. coli and other gram-negative microorganisms
generally cannot secrete proteins into surrounding
medium because of the presence of an outer
membrane.
• To solve the problem, the first is to use gram-positive
prokaryotes or eukaryotic cells as host organisms.
• The second solution entails the use of genetic
engineered gram-negative bacteria that can secrete
proteins directly into growth medium.
• Bacteriocin release factor gene can be co-expressed on
the other plasmid to facilitate the secretion.
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77. Increasing Secretion
Secretion into the Medium
• Although secretion of E. coli proteins is quite rare, the
small protein YebF is naturally secreted to the medium
without lysing the cells or permeabilizing the
membranes.
• When various proteins are fused to the C-terminal end of
YebF, following the removal of the signal peptide, the
entire fusion constructed is secreted to the medium.
• The next step will likely involve engineering a readily
cleavable linker region between YebF and the protein of
interest so it can be recovered in its native form.
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78. Increasing Secretion
Secretion into the Medium
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79. Metabolic Load
• The over-expression of a foreign protein prevents cell
from obtaining sufficient energy and resources for its
growth and metabolism so that it is less able to grow
rapidly and attain high density.
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80. Metabolic Load
• An increasing plasmid copy number and/or size requires
increasing amounts of cellular energy for plasmid
replication and maintenance.
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81. Minimize the Metabolic Load
• The metabolic load can be decreased by using a low-
copy number rather than a high-copy-number plasmid
vector or integration the foreign DNA directly into the
chromosomal DNA of the host organism.
• The use of strong but regulatable promoters is also an
effective means of reducing the metabolic load.
• Completely or partially synthesizing the target gene to
better reflect the codon usage of the host organism.
• Accept a modest level of foreign-gene-expression-
perhaps 5% of the total cell protein-and instead focus on
attaining a high host cell density.
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