Microbial Gene manipulation and

Expression
Sogandi, M. Si
A Gene Encodes for A Protein:
Gene can be Manipulated
MUTATION
 MUTATION
Regulatory mutation
Based on The Structural mutation
Location
Mixed mutation

Base-Structural mutation

?
Type of
Mutation
Point mutation
Substitution, Deletion,
Insertion
Based on The
Change Base-Sequence mutation
Chromosomal mutation
Translocation,
Inversions, Duplication,
Insertion, Deletion
 CHROMOSAL MUTATION

 DUPLICATION
 DELETION
 INVERSION
 TRANSLOCATION
CHROMOSAL DUPLICATION
 Terminal tandem duplications
occur when the duplications
are arranged tandemly at the
end of a chromosome
 Duplications have played an
important role in the
evolution of multigene
families such as the genes for
hemoglobin in humans
 Carcot Marie Tooth Desease,
Down syndrome
CHROMOSAL DELETION

 A deletion is a chromosomal
mutation involving the loss of
a chromosomal segment that
begins where breaks occur.

 Deletion mutations cannot

revert to wild-type.
 CHROMOSAL INVERSION

Chromosome segment breaks off

Segment flips around backwards
Segment reattaches
CHROMOSAL TRANSLOCATION
 Movement of chromosomal segment to non-
homologous chromosome
 Intrachromosomal and Interchromosomal
translocations
 Reciprocal & NonReciprocal translocation
Gene Cloning and Expression
Gene Cloning
Bacteria cannot carry out RNA splicing
Characteristic Host
Choose of Vector
 Increase selectivity of
protein purification:
(Gene fusion strategies)
Most target protein lack a suitable
Affinity ligand usable for capture on
a solid matrix. A way to circumvent this
obstacle is to genetically fuse the gene
encoding the target protein with a gene
encoding a purification tag. When the
chimeric protein is expressed, the tag
allows for specific capture of the fusion
protein. This will allow the purification
of virtually any protein without any prior
knowledge of its biochemical properties.
 Advantages and disadvantages for
using tags in fusion proteins
Plus factors:
(1)improve protein yield (2) prevent proteolysis (3)
facilitate protein refolding (4) protect the antigenicity of
the fusion protein and (5) increase solubility (6) increase
the sensitivity of binding assays for tagged ScFv.

Minus factors:
(1) a change in protein conformation (solubility and
activity) (2) lower protein yields (cleavage may not be
complete) (3) inhibition of enzyme activity (4) alteration
in biological activity (5) undesired flexibility in
structural studies (6) cleavage/removing the fusion
partner requires expensive protease (Factor Xa,
enterokinase) and (7) toxicity.
 Commonly used affinity tag system in
recombinant protein expression:

1. expression and purification of maltose-binding

protein fusions. (provides a factor Xa cleavage
site).
2. expression and purification of Glutathione-S-
transferase fusion proteins. (contains either a
thrombin cleavage site, a factor Xa cleavage site,
or an Asp-Pro acid cleavage site).
3. expression and purification of thioredoxin fusion
proteins. (provides an enterokinase cleavage
site).
4. expression and purification of 6X His-tagged
proteins.
General problems with heterologus gene
expression

(a) Not enough protein is produced:

* codon usage preferential (rare codon)
* potential mRNA secondary structure.(5’-end
ATcontent, 3’-end transcriptional terminator)
* toxic gene.
(b) Enough protein is produced, but it is insoluble:
* vary the growth temperature.
* change fermentation medium.
* low-copy-number plasmas.
* selection of promoter.

The KEY idea is to slow down the expression rate of

protein.
OPTIMIZING TRANSCRIPTION OF THE
CLONED GENE

1. genetic fusion to strong promoters

(transcriptional fusion).
2. increased gene dosage (utilize the gene’s
own promoter with the gene on a high-
copy plasmid).
3. potential problem with toxic genes and
available methods for efficient repression.
4. solutions to potential problems with
premature termination and mRNA
instability.
OPTIMIZING TRANSLATION OF THE CLONED
GENE

1. sequence determinants for translation

initiation (Shine-Dalgarno sequence).
2. translational fusion vectors.
3. potential problem with biased codon
usage.
4. enhancing the stability of protein
products.
Expression Vector
Strong and Weak promoters
Penggunaan Dua Vektor
Optimasi Kodon
Gene Expression
Microbial heterologous expression
systems

Heterologous expression means

that a protein is expressed in a cell
that does not normally make
(express) that protein.
1. Escherichia coli
2. Yeast (Saccharomyces cerevisiae, Pichia pastoris etc )
 Factors affecting expression of
heterologous proteins

1. trans-acting factors and cis-acting elements

2. Affinity tag used
3. Size of the heterologous protein
4. Source of the heterologous protein
5. Codon biasing
6. E. coli host strain used
7. Culture conditions
8. Degradation of the heterologous protein
Size of the heterologous protein

• Proteins less than 100KDa are well tolerated

in E. coli and hence are expressed to
significant levels in the bacterium.

• Proteins more than 100KDa are unable to

express properly in the bacterium and hence
are degraded.

• If the size is exceeding this limit then host

systems other than E. coli should be used.
Source of the heterologous protein

• Many eukaryotic proteins are not

appreciably expressed in E. coli.

• Membrane proteins are also unable to

express properly in E. coli. In order to
express such proteins other expression
systems (yeast, insect or mammalian cell
lines) are used.
 E. coli host strain used
• Choosing the proper E. coli host strain is obligatory
for the substantial expression of recombinant
proteins. It should be protease deficient.

• The most commonly used protease deficient strain is

BL21.

• Certain affinity tags require specific E. coli host

strains. For example, TB1 E. coli host strain is used
to express proteins tagged with maltose binding
protein (MBP).
 Culture conditions

• Optimizing the culture medium (like LB or 2YT) is mandatory

for the expression of heterologous proteins in E. coli.
• Proper aeration should be maintained. Optimum is 20% rule.
• Volume of the culture media should not exceed the 20% of the
maximum capacity of the flask.
• Induction at proper growth phase also affects protein
expression. Mostly, mid log phase cultures are used for
induction.
• Concentration of inducer should also be empirically
determined..
• Proper temperature also affects the expression. Grow the
cultures at 37ºC and after induction grow at 23-30ºC.
Expression of human interferon gene
in bacterial cells

Example :
Expression of human interferon gene
in bacterial cells

Gene isolation
Cloning into Expression Vector
Gene Expression and Protein
Purification
Western Blot Analysis
Thank You