Manipulation of gene Expression

in Bacteria
EXPRESSION

EXPRESSION


EXPRESSION
Biotechnology
The primary objective of gene cloning or
genetic engineering is to express the cloned
gene in a selected host organism
Host System
Bacteria
Yeast
Insect
Mammalian cells
GENETICALLY
ENGINEERED PRODUCTS
Some products produced by genetic
engineering and are currently on the market

Bt potatoes
PRV Papaya
Herbicide resistant corn
Human growth hormone
Insulin
human interferon protein
monoclonal antibodies
Gene expression in different
systems
Bacterial genes being expressed in a
eukaryotic system
Eukaryotic genes are being expressed
in bacteria
Expression vectors
Vectors designed to overproduce
specific gene products
Use well-characterised RNA and protein
synthesis systems

Lambda ZAPII
pTrc99A
pBluescript II SK+


Manipulations to modulate
gene expression
The transcriptional promoter and terminator
sequences
The ribosome-binding site and the efficiency of
translation in the host organism
The number of copies of the cloned gene and
whether the gene is plasmid borne or integrated into
the genome of the host
The intrinsic stability of the cloned gene protein within
the cell
The final cellular location of the synthesized foreign
protein
Promoters
Effective gene expression system
requires a strong and regulatable
promoter sequence upstream from the
cloned gene
Strong promoter has a high affinity for
RNA pol

Regulated promoter
Some cloned DNA may produce products that are
toxic to the bacterial cell, and if the promoter driving
production of such gene products were not regulated,
the bacterial cell might be poisoned.
High levels of continual expression of a cloned gene
is often detrimental to the host cell because it creates
an energy drain, thereby impairing essential host cell
functions.
Plasmids carrying a constitutively expressed gene
may be lost after several generation since cells
without plasmids can take over the culture
What is the best promoter type for You?
Constitutive promoters always
express your gene of interest and
eliminate the extra complexity of adding
an inducer.
If you have a non-toxic gene and are
not worried about the timing of your
expression, using a constitutive
promoter is easier.

Examples of Promoters
T3
T5
T7
Lac
Trp
35S from CMV
Chicken actin

Promoter
The 35S promoter for CMV is used to
express viral genes in plants because a
bacterial promoter or bacteriophage
promoter cannot turn on genes in plants
Mammalian expression uses the strong
constitutive CAG promoter that consists of
the chicken actin promoter

Tri Systems www.Qiagen.com
A single vector is possible in three different
expression systems
the T5 promoter/lac operator transcription-
translation system for expression in E. coli
the p10 promoter for baculovirus-based
expression in insect cells
the CAG (actin) promoter for expression in
mammalian cells
Regulated expression vector
pTrc99A (Ref: Amann et al. Gene 69: 301-315 (1988))
Designed for the regulated expression of genes in
E. coli of fused and non-fused proteins
Based on pKK233-2
Origin of replication from pBR322
Has a strong hybrid promoter trp/lac
The lacZ ribosome binding site (RBS)
MCS of pUC18
rrnB transcription terminator

Ptac or Ptrc promoter
Regulated hybrid promoter containing the 35
promoter from the trp promoter and the 10
region from the lac
uv5
promoter
Lac
uv5
is a variant of the lac promoter that contains
altered nt seq in the 10 region is stronger than
the wild type lac promoter and is repressed by the
lac repressor and derepressed by IPTG or lactose
Does not require activation with CAP-cAMP

Lac
In the absence of lactose, the E. coli lac
promoter is repressed, turned off by the lac
repressor protein
Induction can be achieved by the addition of
lactose or IPTG, prevents the binding of the
repressor to the lac operator
Catabolite activator protein (CAP) increases
affinity of the promoter for RNA pol
Trp
Trp promoter is negatively regulated, turned
off by a tryptophan trp-repressor protein
complex that binds to the trp operator and
prevents transcription of the trp operon
De repression, or turning on achieved by
removing tryptophan or adding 3-indole
acrylic acid
Consensus sequences
5-TATAAT-3 for the 10 region
5-TTGACA-3 for the 35 region

The lacUV5 promoter has a consensus
sequence at its 10 but not its 35 region
The trp promoter has a consensus sequence
at its 35 region but not its 10 region

By fusing the 10 region of the lac promoter and
the 35 region of the trp promoter, called it the
Ptac or Ptrc
pTrc99A
lacI gene including its corresponding
promoter (P
lacI
).
Also carries the lacI
q
mutation for which
helps to maintain repression of P
lac

transcription.
Produces much higher levels of the lac
repressor, thereby decreasing the leakiness
under non-induced conditions, i.e.
transcription of a cloned gene in the absence
of an inducer
pTrc99A
To produce the protein in pTrc99A, cells
carrying the plasmid are grown to a
moderate density and IPTG is added
Increasing protein production
Strategy used to increase protein production
in recombinant E. coli involves designing an
expression vector with a Ts origin of
replication and a regulated promoter
Replacing the origin of replication of the plasmid
pPLc2833 with that from the plasmid pKN402
To make the plasmid pCP3
pCP3 contains the p
L
promoter, the -lactamase
gene for amp resistance from pLc2833 and the
origin of replication from pKN402
cI repressor is present in the chromosome of the E.
coli host
The p
L
promoter is controlled by the cI repressor
protein of the bacteriophage lambda, this switches
off the transcription from p
L
promoter
Cells that carry the pCP3 plasmid are grown first
at 28C and then shifted to 42C
At 28C the cI repressor is functional and turns off
the p
L
promoter plasmid copy number is normal
At 42C, the plasmid copy number increases and
the ts cI repressor is inactivated
Large scale production systems
Temperature shifts take time and energy
Chemical inducer e.g. IPTG is expensive
Large scale production systems
Two Plasmid system:

In one plasmid:
The cI repressor was placed under the trp
promoter (Ptrp) and inserted into a low copy
plasmid
In a second plasmid:
Have the cloned genes, e.g. -galactosidase and
citrate synthase genes under the p
L
promoter
Termination signals
Two strong transcription terminators T7
from E. coli bacteriophage T7 and rabbit
globin terminator sequence
Contains signals for the polyadenylation of
the mRNA transcript to prevent read-
through transcription to ensure stability of
the expressed construct

Regulatory sequences
Ribosome binding site (RBS)
The molecular basis for differential
translation is the presence of a
translational initiation signal called a
ribosome binding site (rbs) in the
transcribed mRNA

RBS
Bacterial cells and human cells have
different mechanisms for selecting
translational start points on the mRMA
This is an important consideration for
producing human proteins in bacteria
RBS- Bacteria
Directed by a ribosome attachment sequence
closely linked to the AUG initiator codon for
translation.
This ribosome attachment sequence consist
of 6 8 nucleotides UAAGGAGG (Shine
Dalgarno sequence) upstream of AUG
This sequence is complementary to the 3
end of the 16S ribosomal RNA, AUUCCUCC
RBS
Activity of a RBS can be influenced by the length
and nucleotide composition of the spacer
separating the RBS and the initiator AUG
Bacterial mRNAs that do not have a close match
to the consensus ribosome attachment sequence
are not translated efficiently
Generally, the stronger the binding of the mRNA
to the ribosomal RNA, the greater the efficiency
of translational initiation

RBS - Eukaryotes
The ribosome binds to an mRNA
through the 5 terminal cap structure
The ribosome then scans the RNA in
the 5 to 3 direction until it located the
first AUG in the mRNA
If this AUG is surrounded by a suitable
sequence, translation will begin

RBS - Eukaryotes
This sequence in eukaryotes, called the
Kozak sequence 5-A/GCCACCAUGG
which lies within a short 5' untranslated
region, directs translation of mRNA

Conditions required for maximum
translation efficiency
The RBS must be located at a precise distance
from the translation start codon of the cloned gene
Overproducing a human protein in E. coli requires
customizing the gene to have a bacterial RBS
appropriately spaced from the AUG codon for
translation initiation
The DNA sequence that includes the RBS through
the first few codons of the gene must not contain
nucleotide sequences that after transcription can
fold back to form intra-strand loops, thereby
blocking the interaction of the mRNA with the
ribosome.

Expression vector pKK233-2
An ampicillin resistance gene
The tac promoter
The lacZ ribosome binding site
An ATG start codon located 8
nucleotides downstream from the RBS
The transcription terminators T1 and T2
from lambda
Copies of the cloned gene
Tandem gene arrays
level of gene expression is proportional to
the number of copies of the transcribed
gene in the host cell
STRATEGY
Increase plasmid copy in the cell
Clone multiple copies of the gene in tandem
into a low copy number plasmid
Vector Preparation
Unique site in vector is digested with AvaI
(C_TCGGG)
Filling in with DNA polymerase I
EcoRI linker (GAATT_C) inserted
EcoRI linker flanked by two AvaI sites
(CTCGGAATTCTCGGG) is inserted into the
plasmid
The gene plus the signals are cloned into the
EcoRI site by digesting with AvaI to give non-
sticky ends so the genes are orientated in
one direction
DNA integration into the host
chromosome
When a gene is part of the host
chromosomal DNA, it is relatively stable
and consequently can be maintained for
many generations
Factors to consider when
integrating a gene
the chromosome integration site must not be
within an essential coding gene
the input gene must be under the control of a
regulated promoter
for integration the DNA sequence must have
some sequence homology for recombination
between the two DNA molecules
chromosomal site about 50 nt


Integration into the host
chromosome
The cloned gene inserted in the middle of a
cloned segment of DNA (ab) from the host
chromosome on the plasmid
Homologous DNA pairing occurs between
plasmid-bourne DNA regions a and b and the
host chromosome DNA regions a and b
A double cross over event (X-X) results in the
integration of the cloned gene
Stability of cloned gene
protein
Small proteins and peptides are difficult to
be stably expressed in E. coli
These proteins can be stabilised by
expressing them fused to a large protein
such as mouse (DHFR) dihydrofolate
reductase
Fusion Proteins
Fusion proteins are constructed at the DNA
level by ligating together the coding regions
of two genes
One gene is the host gene that produces a
stable host protein and the other gene
represents the cloned gene that will produce
a foreign protein
When the two genes are transcribed, the
foreign protein produced will be covalently
linked to the stable host protein

Uses of fusion protein
determine its location in a cell
stability of the protein
used as antigens and to generate
antibodies
Used to purify recombinant proteins
through the technique of immuno-affinity
chromatography
Histidine tag
Fusion proteins can be designed with a His
tag
Can be placed at either N- or C- terminus
Important that DNA is inserted in correct
reading frame
His tag
His tag at the N-terminus most commonly used,
easiest to prepare
When at the N-terminus only the 5end of the
ORF must be ligated in frame
When at the C-terminus the insert must be cloned
in frame with the ATG start codon and the 3 His
coding system
At the C-terminus His tag facilitate cloning of full
length

Cleavage of fusion proteins
Host protein

may affect the biological functioning of the
target protein
may cause allergic responses
Marker Peptide
Saccharomyces cerivisiae plasmid
construct for the production of
interleukin-2 is joined to DNA encoding
a marker peptide sequence
(Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys)
Function of Marker Peptide
reduces degradation of the expressed
interlukin-2 gene product and
enables the product to be purified

Purify recombinant proteins
Monoclonal antibodies made against the
marker peptide
Immobilized on a polypropylene support
and binds the fusion proteins marker protein
The secreted proteins are passed through the
column containing the bound antibody
The immunopurified fusion protein is then
selectively eluted from the column
marker protein is removed with bovine
intestinal enterokinase.
Some Fusion Protein System
Fusion Partner Size Ligand Elution Condition
Protein A 14kb IgG Low pH
His tail 6-10aa Ni2+ Imidazole
Strep-tag 10 aa Streptavidin Iminobiotin


Metabolic load
Increasing plasmid copy number or
plasmid size
Limited amounts of dissolved oxygen in
the growth medium
Overproduction of foreign proteins
Jamming of export site
Production of toxic products
Overcoming O
2
Limitation
Use of protease-deficient strains
develop host strains that are deficient in the
production of proteolytic enzymes
mutations in both the genes for the RNA pol
sigma factor responsible for heat shock proteins
synthesis (rpoH) and the gene for protease
required for growth at high temperatures (degP)
secreted proteins had a 36-fold greater specific
activity
Overcoming O
2
Limitation
Bacterial hemoglobin
Vitreoscilla bacterium, GNB obligate aerobe,
normally live in oxygen poor environments
such as stagnant water.
synthesizes a hemoglobin-like molecule
gene for Hb-like protein cloned and expressed in
E. coli, the transformant displayed higher levels of
protein synthesis
Overproduction of foreign
proteins
deplete the pools of certain aminoacyl-
tRNA or even certain amino acids.
increase translational errors 10-fold
drain the host cell of it energy
diminish the usefulness of the protein
may cause the protein to be
immunogenic
Secretion of proteins
Secretion of proteins in E. coli is mediated
by an N-terminal signal sequence
Promote proper folding and disulfide bond
formation
Direct toxic proteins out of the cell
Jamming of export site
Over expression of protein and export from
cytoplasm to cell membrane or periplasm
may jam export sites thereby preventing
localization of essential proteins
Over expressed protein may also interfere
with proper functioning of the host cell
Use of His-tag at the C-terminus to prevent
interferences during N-terminal processing
Reference
Glick and Pasternak. Molecular
Biotechnology: Principles and Applications
of Recombinant DNA 2
nd
Edition
Chapter 6.