Microbial Biotechnology
Lec. 8
Manipulation of Gene Expression
in Prokaryotes
Dr. Marwan Abu-Halaweh
Office 908
Email:mhalaweh@philadelphia.edu.jo
Microbial Biotechnology
Philadelphia University
Fusion Protein
• Foreign protein especially small ones are
produces in small amount due to their
degradation.
• One way to solve this problem is to engineer a
DNA construct that encode target protein that
is in frame with a stable host protein.
• This combined single protein (fusion protein),
protects the cloned gene product from attack
by host proteases.
Cleavage of Fusion proteins
• The presence of host protein make most protein not
suitable for clinical use.
• Also target protein needs to many tests before it is
become available for comercial use.
• Thus there is a need to cleaved the target protein from
host one.
• This can be done at gene level by add a tail of specific
amino acids or proteins that code for proteases
recognition site.
• For example a DNA linker coded for Ile-Glu-Gly-Arg aa
can be added to the fusion protein gene. After protein
synthesis coagulation factor called Xa (specific for
proteases recognition site) could be used.
Uses of Fusion Proteins
• For some application fusion protein can be
satisfactory end products.
• For example a specific antigen (stabilizing protein)
site that is required in large amount and is a part of
the fusion protein may be used for research or
diagnosis as long as the stabilizing protein dose not
interfere with the correct folding of antigen
recognition site.
• In this case the fusion protein can be use as an antigen
and any antibodies that recognize the stabilizing
protein, can be removed or detected.
Interleukin-2
1- Concentrate secreted
protein mixture
Immunoaffinity
chromatography
2- Prepare
purification of a fusion
Immunoaffinity
column
protein.
Antibody (Ab) bind to
markers peptide of fusion 3- Added secreted
protein is attached to a solid protein mixture to
the column
polypropylene support.
Marker peptide
Polypropylen
e supporrt
Anti-Marker peptide
Ab
Marker peptide binds to Ab
4- Elute fusion protein
Other protein pass
through the colum
Fusion protein
Marker peptide
Interleukin-2
Increasing Protein Stability
• Under the normal growing
conditions, the half-lives of
different protein ranges
from a few minutes to
hours.
• The basis for this
differential stability is both
extended of disulfide bond
formation and the presence
of certain amino acid at the
N-terminus.
Use protease deficient host strains
• One way to stabilize foreign protein is to develop
host strains that are deficient in the production of
proteolytic enzymes.
• This is a very complex procedure, for example E.
coli has at least 25 different proteases, and few is
studied at gene level.
• These proteases are important for the degradation of
abnormal or defective protein, which is known as
housekeeping function that is necessary for viability
of the cell.
• E. coli with mutation in both gene for the RNA
polymerase sigma factor that is responsible for heat
shock protein synthesis and the gene for protease that
is required for cell growth at high temperatures,
secreted protein had a 36-fold greater specific activity
than they were produced in wild-type host.
Express bacterial hemoglobin in E. coli
• Some species like Vitreoscilla bacterium, which is a G-ve
bacteria synthesize a haemoglobin like molecules that binds
oxygen from the environment and increase the level of oxygen
available inside cells.
• when this gene transfer into E. coli the transformants
displayed:
– a higher levels of proteins synthesis of both cellular and
recombinants proteins.
– More efficient proton pumping
– A higher ATP production
– A higher ATP contents.
DNA Integration into the Host
Chromosome
• Normally plasmid increase cell load because of the
energy that used for its replication, transcription and
translation.
• As a result a fraction of the cell population loses its
plasmid during cell growth.
• Also cell lack plasmid grow faster.
• To solve this problem the cell can grow in a medium
that contain certain antibiotics or essential metabolite
that only coded by plasmids.
• This solution could be costly at large scale
fermentation.
DNA Integration into the Host
Chromosome
• The other way to solve the plasmid loss is to clone the
DNA directly into the chromosomal DNA of the host.
• Which make this DNA fragment more stable and
consequently can be maintained for many generation in
the absence of selective agent.
• The integration must not be within the essential coding
gene.
• This can be done by targeting non essential site within the
chromosome.
• Also input protein should be under the control of
regulatable promoter.
DNA Integration into the Host
Chromosome
• For integration of the DNA into chromosomal
site, the input DNA must share some
sequence similarity at least 50 nucleotides,
with the chromosomal DNA, those nucleotide
should recombined with the match sequence
within the chromosome.
• This could be done according to the following
protocol.
Protocol for DNA Integration into
the Host Chromosome
1. Identify the desired chromosome integration site.
2. Isolate and clone part of the chromosomal
integration site.
3. Ligate a cloned gene and regulatable promoter either
into or adjacent the cloned chromosomal integration
site.
4. Transfer the chromosomal integration fragmentcloned gene construct into the host cell as part of
plasmid that can not replicate into the host cell.
5. Select and propagate the host cell that express the
cloned gene.
DNA Integration into the Host
Chromosome
• When host cell is transformed with a non
replicating plasmid that carries the cloned
gene in the middle of the cloned
chromosomal integration site, then the
DNA in the plasmid can base pair with
identical sequence on the host
chromosome.