Transcript 2. Genetic methods

Protein Biotechnology
Protein Biotech
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Construct design for high-level transgene
expression in animal cells
When ever a recombinant protein is needed to be produced
the ultimate goal will be the production of the maximum
yield. Therefore, the following considerations should apply
when a high level of expression is required:
 The use of strong and constitutive promoter
 The inclusion of an intron
 The inclusion of a polyadenylation signal
 The removal of unnecessary untranslated sequence
 Optimization of the transgene for translational efficiency
 The incorporation of a targeting signal
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The use of strong and constitutive
promoter
Very active promoters produce the highest level of
transgene expression. In viral systems, the transgenes
are expressed under the strongest endogenous
promoter such as;



Baculovirus polyhedrin promoter
Adenoviral E1 promoter
Vaccinia virus p 7.5 promoter
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The inclusion of an Intron
The presence of an intron in a eukaryotic expression
system usually enhances expression. Currently most
mammalian expression systems incorporate a heterologous
intron such as the SV40 small t- antigen intron or the
human growth hormone intron. However, introns may not
by used in some expression systems such as vaccinia virus.
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The inclusion of a polyadenylation signal
Polyadenylation signals (terminators) are required in eukaryotic
genes to generate a defined 3’ end of the mRNA.
This is normally done by the addition of several hundred
adenosine residues to generate a poly A tail. This tail is
required for the export of mRNA into the cytoplasm and to
increase its stability.
It is found that in the absence of the 3’ end with the poly A sites
of the mRNA, the level of expressed protein fall down by as
much as 90%.
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The inclusion of a polyadenylation
signal… cont.
Example; the poly A site from the SV40 transcription unit or
the mouse beta globin gene are often incorporated into
mammalian expression vectors to enhance expression.
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The removal of unnecessary untranslated sequence
Eukaryotic mRNAs comprise a coding sequence (gene) bracketed by untranslated
regions (UTRs) of variable lengths. Both the 5’ and 3’ UTRs can influence gene
expression in a number of ways;
5’ UTR may contain one or more AUG codons upstream of the
authentic translational start site. Such codons are detrimental to the
translation initiation.

The 3’ UTR may contain regulatory elements (AU-rich) that control
mRNA stability.

Both the 5’ and 3’ UTRs may be rich in secondary structure that
prevents efficient translation. Therefore, UTR sequences are generically
removed from transgene construct to maximize expression

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Optimization of the transgene for
translational efficiency
The sequence around the translational initiation site should conform to
Kozak’s consensus, which is defined as 5’-CCRCCAUGG-3’.

Of great importance is the purine at the -3 position ® and the
guanidine at the +4 position. The adinosine in the AUG initiation codon is
given the number +1 and the immediately preceding base is defined as 1.

The expression of foreign genes in animals can be inefficient in some
cases due to suboptimal codon choice. i.e different organisms prefer to
use different codons to specify the same amino acid. Therefore, if a
transgene contains codon that is commonly used in the source organism
but rarely used in the host, the translation may pause at that codon due
to the scarcity of the corresponding tRNA. This will reduce level of
protein synthesis or may even lead to the truncation of the protein or
frame shifting.

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The incorporation of a targeting signal
If the goal of the expression is to produce large amount of
functional protein, then it is necessary to consider whether
it should be post-translationally modified or not.
Example glycosylation might be needed for therapeutic
proteins. This is important for the correct function of the
protein and to minimize the immune response against it.
It is important to target the protein to the correct
compartment to be appropriately modified. This can be
done by incorporating a signal peptide if the protein that
has to be glycosylated.
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Many mammalian expression vectors are available for this
purpose.
Example, the Invitrogen vector pSecTag2 which incorporates a
sequence encoding the murine immunoglobulin light chain
signal peptide for targeting the produced protein to the
secretory pathway.
In addition, the C terminus of the recombinant protein is
expressed as a fusion protein to two different tags to facilitate
its purification.
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Purification of a cloned gene product can
be facilitated by use of purification tags
Many cloning vectors have been engineered so that the protein being
expressed is fused to another protein called tag to facilitate its purification.
Examples of such tags are;
glutathione S-transferase
 MalE (maltose binding) protein
 Multiple histidine residues (his tag) which can be easily purified by metal
affinity chromatography.

The vectors are usually constructed so that the coding sequence for an
amino acid which is cleaved by specific protease is incorporated between
the coding for the tag and the gene being expressed.
It is also possible to include with the tag a protein that can be assayed
easily to facilitate detection of the protein.
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Examples of the tags
The use of polyhistidine fusion for purification;
the gene of interest is first engineered into a vector in which there is a
polylinker downstream of six histidine residues and a proteolytic
cleavage site which could be a site for enterokinase.
1.
After induction of synthesis of the fusion protein, the cells are lysed
and the viscosity of the lysate is reduced by neuclease treatment.
The lysate is then applied to the column containing immobilized
divalent nickel that selectively binds to the polyhistidine tag.
After washing away any none specific material, the fusion protein is
eluted from the column and will be treated with enterokinase to
release the cloned gene product.
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To facilitate the assay of the fusion protein, short antibody
recognition sequences can be incorporated in the tag
between the affinity label and the protease cleavage
site.
Example of these sequences:
1.
Glu-Gln-…………Asp-LeuAnti-myc antibody
2.
His-His-His-His-His-His-COOH
Anti-His (Cterminal) antibody
3.
Gly-Lys-………………Asp-Ser-ThrAnti-V5 antibody
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An example of a purification tag is the incorporation of biotin
carboxylase carrier protein.
A protein that has the biotin tag can be purified by a
streptavidin affinity column.
E. coli expresses a single endogenous biotinylated protein but
it does not bind to streptavidin in its native conformation
making the affinity purification highly specific for the
recombinant fusion proteins. In addition, the biotin on the
fusion protein can be detected by enzymes coupled to
streptavidin.
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Vectors that promote solubilization of
expressed proteins
One of the problems associated with production of over
expressed proteins is the formation of the inclusion
bodies. How to get rid of the problem?
1.
Culture methods such as reducing the temperature and
changing media composition and pH values to reduce
growth rate can minimize the problem. Or growing
bacteria under osmotic stress (4% NaCl + 0.5 M sorbitol )
in addition, adding glycine betaine which protects
proteins at high salt concentrations lead to generation of
correctly folded proteins.
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2.
Genetic methods;
1.
Host cell is engineered to overproduce chaperons + the protein (even
though there is no guarantee of proper folding of the protein).
2. Making minor changes to the amino acid sequence of the target protein
e.g cysteine-to-serine changes in fibroblast growth factor minimized
inclusion-body formation.
3.
Synthesis of target proteins (who aggregates in their native form) as
thioredoxin fusion proteins or as maltose-binding protein or as fusion
with some other proteins such as NusA, GrpE proteins or
bacterioferritin. Enterokinase Cleavage site is incorporated to facilitate
the purification of these fusion proteins later.
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1.
An alternative way to keep proteins soluble is to export
them to the periplasmic space
Ps. After synthesis, the fusion protein is
released by osmotic shock and purified, then
cleaved by the enterokinase to release the
target protein.
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Exporting proteins to periplasmic space
In E. coli protein export to the periplasmic space or to the
outer membrane is achieved by a universal mechanism
known as the General Export Pathway (GEP).
Proteins that enter the GEP are synthesized in the
cytoplasm with a signal sequence at the N-terminus.
 This sequence is cleaved by a signal or leader peptidase
during transport.
 The signal sequence has three domains; +ve charged
amino terminal region, hydrophobic core and a leader
peptidase cleavage site.

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Exporting proteins to periplasmic space..
Cont.
A signal sequence attached to a normally cytoplasmic
protein will direct it to the export pathway.

Many signal sequences that are derived from naturally
occurring secretory proteins (ompA, ompT, AP… etc)
support the efficient translocation of heterologous peptides
across the inner membrane when fused to their amino
terminus.

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Optimizing Translation
High level expression of genes requires more than a strong
promoter. There are many factors that can influence the
rate of translation:
1.
The interaction of the ribosome with bases immediately
upstream from the initiation codon of the gene (most
important).
In bacteria a key sequence is the ribosome binding site or
Shine-Dalgarno (S-D) sequence.
2.
The degree of complementarity of this sequence with
the 16S rRNA can affect the rate of translation.
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Optimizing Translation …..con.
The spacing between the S-D sequence and the initiation codon is
also important. Usually there are 5 to 10 basis with 8 being
optimal. Decreasing the distance below 4 or increasing it above 14
can reduce translation several fold.
3.
Translation can also be affected by sequence following the S-D site.
The presence of 4 A or 4 T residues in this position gave the
highest translation efficiency while the translational efficiency
decreased to 50 or 25% when 4 C or 4 G residues where present.
4.
5.
The composition of the triplet immediately proceeding the AUG
start codon or the composition following the AUG also affects the
efficiency of translation. Highly expressed genes have AAA (Lys) or
GCU (Ala) as the second codons.
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Optimizing Translation…. Cont.
6. Sequence upstream from the S-D can also affect the translation of
certain genes. e.g in E. coli md gene there is a run of 8 uracil
residues. Changing two to 5 of these residues had no effect on
mRNA level, but reduces translation by 95%.
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End of chapter
Protein Biotech
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Protein Biotech
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Protein Biotech
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Protein Biotech
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Protein Biotech
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