Transcript Chapter 6

Manipulation of Gene
Expression in Prokaryotes
Gene Expression in Prokaryotes
• The expression of the cloned gene in a selected
host organism.
• It does not necessarily ensure that it will be
successfully expressed.
• A high rate of production of the protein encoded
by the cloned gene is required.
• Specialized expression vectors have been
created that provide genetic elements for
controlling transcription, translation, protein
stability, and secretion of the product from the
host cell.
Manipulation of Gene Expression
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The promoter and transcription terminator sequences.
The strength of the ribosome-binding site.
The number of copies of the cloned gene.
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.
• The intrinsic stability within the host cell of the protein
encoded by the cloned gene.
Gene Expression from Strong and
Regulatable Promoter
• The strong promoter is one that has high affinity for
RNA polymerase that the adjacent downstream region
is frequently transcribed.
• The ability to regulate a promoter enables the cell to
control the extent of transcription in a precise manner.
• The lac operon of E. coli has been used extensively for
expressing cloned genes.
• Many different promoters with distinctive properties
have been isolated from a range of organisms.
Gene Expression from Strong and
Regulatable Promoter
• The most widely used are those from the E. coli lac
and trp operons.
• The tac promoter is constructed from -10 region of the
lac promoter and -35 region of the trp promoter.
• The pL promoter from bacteriophage λ.
• The gene 10 promoter from bacteriophage T7.
• Each of these promoters interacts with regulatory
proteins which provide a controlable switch for either
turning on or turning off specific transcription of
adjacent cloned gene.
Gene Expression from Strong and
Regulatable Promoter
Gene Expression from Strong and
Regulatable Promoter
Gene Expression from Strong and
Regulatable Promoter
• The pL promoter is controlled by the cI repressor
protein from bacteriophage λ.
• A temperature-sensitive mutant of the cI repressor,
cI857, is generally used to regulate pL-directed
transcription.
• Cells are first grown at 28 - 30ºC, at which the cI
repressor prevent transcription.
• When the cell culture reaches the desired stage of
growth, the temperature is shifted to 42ºC.
• The cI repressor is inactivated and transcription can
proceed.
Gene Expression from Strong and
Regulatable Promoter
• The gene 10 promoter from bacteriophage T7 requires
T7 RNA polymerase for transcription.
• T7 RNA polymerase gene is inserted in the E. coli
chromosome on a bacteriophage T7 lysogen under the
control of the E. coli lac promoter.
• In the absence of IPTG, the lac repressor represses
the synthesis of T7 RNA polymerase. Therefore, the
target gene is not transcribed.
• When lactose or IPTG is added to the medium, it binds
to the lac repressor. T7 RNA polymerase is transcribed
and the target gene is transcribed.
• When the portion of the spacer region from the E. coli
lac promoter was mutated, the activity increased >40
fold in the absence of CRP.
• The -20 to -13 region was altered from GC-rich to an
AT-rich.
Increasing Protein Production
• Plasmid pCP3 was created in an effort to obtain the
highest possible level of foreign-protein production in
a recombinant E. coli strain.
• It contains the strong pL promoter, ampicillin resistance
gene, a multiple cloning sequence immediately
downstream from the promoter, and a temperaturesensitive origin of replication.
• The plasmid’s copy number increases 5- to 10- fold
when the temperature is increased to 42ºC.
• At lower temperature, the cI repressor, integrated to E.
coli chromosomal DNA, is functional. The pL promoter
is turned off, and the plasmid copy number is normal.
• At higher temperature, the cI repressor is inactivated,
the pL promoter is active, and the plasmid copy number
increases to around 600 copies per cell, increasing
protein production.
Large-Scale Systems
• In large-scale systems, shifting temperature requires
time and energy, both of which can be costly.
• The cost of chemical inducer, such as IPTG, make the
overall process uneconomical.
• To overcome some of the problems, a two-plasmid
system has been developed.
• The cI repressor was placed under the control of the
trp promoter and inserted into a low-copy-number
plasmid, ensuring that excess cI repressor molecules
are not produced.
Large-Scale Systems
• Cell can be grown on an inexpensive medium
consisting of molasses and casein hydrolysate, which
contain small amount of free trptophan.
• The cloned gene can be induced by the addition of
tryptone to the medium, which contains enough
trptophan for efficient induction of transcription.
• With no trptophan, the cI repressor is produced and
thereby blocking the transcription of the cloned gene.
• With tryptophan, the cI repressor is repressed, and the
cloned gene is transcribed and translated.
• Promoters that are induced when cells enter stationary
phase may be useful in the design of expression
vectors that are useful for large-scale application.
• The house keeping RNA polymerase sigma factor, σD,
and stationary-phase sigma factor, σS, recognize a
similar sequence.
• The cells would be grown to a high density, as the cells
entered the stationary phase, gene expression would
be induced.
Expression in Other Microorganisms
• Cell can be grown on an inexpensive medium
consisting of molasses and casein hydrolysate, which
contain small amount of free trptophan.
• The cloned gene can be induced by the addition of
tryptone to the medium, which contains enough
trptophan for efficient induction of transcription.
• With no trptophan, the cI repressor is produced and
thereby blocking the transcription of the cloned gene.
Expression in Other Microorganisms
Fusion Proteins
• Foreign proteins, especially small ones, often are
degraded in the host cell.
• A DNA construct that encodes a target protein is in
frame with stable host protein.
• Fusion protein protects cloned gene product from
attack by host cell proteases.
• Fusion proteins are stable because the target proteins
are fused with proteins that are not especially
susceptible to proteolysis.
Fusion Proteins for Purification
Fusion Proteins for Purification
• The target protein is fused to the marker peptide.
• The secrete protein can be purified in a single step by
immunoaffinity chromatography.
• The marker peptide is relatively small, it will not
interfere with the production of the target protein.
Immunoaffinity
chromatography
purification of a
fusion protein
Fusion Proteins for Purification
• As an alternative, it has become popular to generate a
fusion protein containing six or eight histidine residue
attached to either N- or C- terminal end of the target
protein.
• The histidine-tagged protein and other cellular proteins
are then passed over an affinity column of nickelnitrilotriacetic acid that bound to histidine-tagged
protein.
• The elution of the bound protein is done by adding of
imidazole (the side chain of histidine.)
Cleavage of Fusion Proteins
• The marker protein may be undesirable. Thus,
strategies have been developed to remove unwanted
amino acid sequence from the target protein.
• Specific sequence that encode short stretches of
amino acids that are recognized by a specific
nonbacterial protease.
Cleavage of Fusion Proteins
• The most commonly used proteases are enterokinase,
tobacco etch virus protease, thrombin, and factor Xa.
• However, it is necessary to perform additional
purification steps in order to separate both the
protease and the fusion protein from the protein of
interest.
• The protease may cleave the protein of interest at
unintended sites.
• The cleavage reaction may not go to completion.
• To avoid the problems, the self-splicing inteins is used.
• Purification of a protein of interest from an inteincontaining fusion protein bound to a chitin
chromatography column through a chitin-binding
domain.
• Cleavages occurs upon the addition of dithiothreitol.
Surface Display
• The expressed target protein is fused to bacteriophage
M13 protein pIII near its N terminus.
• After M13 replication in E. coli cells, the plaques are
assayed immunologically using antibodies that detect
the present of target protein.
• This is an extremely powerful selection system to find
cDNAs for very rarely expressed but important
proteins.
Surface Display
• 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.
Translation Expression Vectors
• Putting cloned gene under the control of a regulatable
and strong promoter may not be sufficient to
maximize the yield of the cloned gene product.
• The efficiency of translation and the stability of the
newly synthesized cloned-gene protein, may also
affect the amount of product.
• The stronger binding of the mRNA to the ribosomal
RNA, the greater the efficiency of translational
initiation.
• The expression vectors have been designed to
ensure that the mRNA of a cloned gene contains a
strong ribosome-binding site.
Translation Expression Vectors
• For each cloned gene, it is important
to establish that the ribosomebinding site is properly placed and
that the secondary structure of the
mRNA does not prevent its access to
the ribosome.
• AUG – start codon
• GGGGG – ribosome-binding site
• G can also pair with U
The Expression Vectors pKK233-2
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Ampicillin resistance gene as a selectable marker.
ptac regulatable promoter
Ribosome binding site
Cloning site (NcoI, PstI, and HindIII)
Start codon and transcription terminators.
Translation Expression Vectors
• Codon usage might interfere with efficient translation
when a clone gene has codons that are rarely used
by the host cells.
• The host cell may not produce enough of the tRNAs
that recognize these rarely used codons, and the
yield of cloned-gene protein is much lower than
expected.
• An insufficient supply of certain tRNAs may lead to
either a reduction in the amount of the protein
synthesized or the incorporation of incorrect amino
acids into the protein.
Translation Expression Vectors
• There are several approached to alleviate this
problem.
• If the target gene is eukaryote, it may be cloned and
expressed in an eukaryotic host cell.
• A new version of the target gene containing codons
that are commonly used by the host cell may be
chemically synthesized (codon optimization.)
• A host cell that has been engineered to over express
several rare tRNAs may be employed.
Increasing Protein Stability
Intrinsic Protein Stability
• Under normal growing conditions, the half-lives of
different proteins range from a few minutes to hours.
• The presence of a single extra amino acid at the Nterminal end is sufficient to stabilize a target protein.
• Long-lived proteins can accumulate in cells and
thereby increase the yield of the product.
• This phenomenon occurs in both prokaryotes and
eukaryotes.
Increasing Protein Stability
Intrinsic Protein Stability
Increasing Protein Stability
Intrinsic Protein Stability
• In contrast, there are internal amino acid sequences
that make a protein more susceptible to proteolytic
degradation.
• This region is called PEST sequence (proline,
glutamine, serine, and threonine.)
• They are often flanked by clusters of positively
charged amino acid, such as lysine and arginine.
• This may act to mark proteins for degradation within
the cell.
Increasing Protein Stability
Facilitating Protein Folding
• 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.
• Fusion proteins that contain thioredoxin as the fusion
partner remain soluble.
• The target gene is cloned into a multiple cloning site
just downstream from the thioredoxin gene.
Increasing Protein Stability
Facilitating Protein Folding
• Without tryptophan, cI repressor is synthesized to
prevent transcription from the pL promoter and
therefore prevent the production of fusion protein.
• When tryptophan is added, the trp promoter is turned
off, the cI repressor is not synthesized, allowing
transcription and translation of the fusion protein.
• The soluble fusion protein is selectively released by
osmotic shock from E. coli cells into growth medium.
• The native form of protein can be released by
treatment with the enzyme enterokinase.
Increasing Protein Stability
Facilitating Protein Folding
Increasing Protein Stability
Facilitating Protein Folding
• Foreign proteins that contain three or more disulfides
generally do not fold correctly in bacteria and often
form inclusion bodies.
• The gene coded for human tissue plasminogen
activator was coexpressed with gene for either rat or
yeast protein disulfide isomerase to assist protein
folding. However, it did not affect the amount of the
protein that could be obtained.
• Overproduction of DscB results in correctly folded
and active human tPA.
Increasing Protein Stability
Facilitating Protein Folding
Increasing Protein Stability
Coexpression Strategies
• The expression of foreign proteins in E. coli results in the
formation of inclusion bodies of inactive proteins.
• Cultivation of recombinant strains at low temperatures,
resulting improper protein folding, often significantly
increases the amount of active protein.
• However, E. coli grow very slow at low temperatures.
• Recombinant strain of E. coli containing the chaperonin
60 gene and cochaperonin 10 gene can grow at 4 -10ºC.
• However, this is the first step of expressing system for
temperature sensitive proteins.
Overcoming Oxygen Limitation
Protease-Deficient Host Strains
• One possible way to stabilize foreign proteins produced in
E. coli is to use host strains that are deficient in the
production 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.
Overcoming Oxygen Limitation
Bacterial Hemoglobin
• 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.
Overcoming Oxygen Limitation
Bacterial Hemoglobin
Limiting Biofilm Formation
• 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.
• Pili are required for initial attachment of bacteria cell to
solid surface, curli are need for cell-cell and cell-surface
attachment, and colanic acid contributes to the three
dimensional structure of biofilm.
• When genes involved in these three functions are
deleted, the strain of E. coli was unable to form biofilms.
• Transformed bacteria are sensitive to antibiotics and
produce a higher level of recombinant protein.
DNA integration into Host Chromosome
• 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.
DNA integration into Host Chromosome
• 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.
• The chromosomal integration site of a cloned gene must
not be within an essential coding gene.
• The input DNA sequence must be targeted to a specific
nonessential site within the chromosome.
• The input gene should be under the control of a
regulatable promoter.
• 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.
DNA integration into Host Chromosome
Removal Selectable Marker Genes
• 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.
Cre-loxP recombination system
Increasing Secretion
Secretion into the 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.
Increasing Secretion
Secretion into the Periplasm
Increasing Secretion
Secretion into the Periplasm
• The signal peptide at the N-terminal end facilitates its
export by enabling the protein to pass through the cell
membrane.
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.
Increasing Secretion
Secretion into the Periplasm
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.
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.
Increasing Secretion
Secretion into the Medium
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.
Metabolic Load
• An increasing plasmid copy number and/or size requires
increasing amounts of cellular energy for plasmid
replication and maintenance.
Minimize the Metabolic Load
• The metabolic load can be decreased by using a lowcopy 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-expressionperhaps 5% of the total cell protein-and instead focus on
attaining a high host cell density.