Microbial Biotechnology Lec. 8

Manipulation of Gene Expression in Prokaryotes

Dr. Marwan Abu-HalawehOffice 908

Email:mhalaweh@philadelphia.edu.joMicrobial BiotechnologyPhiladelphia 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.

Immunoaffinity chromatography purification of a fusion protein.Antibody (Ab) bind to markers peptide of fusion protein is attached to a solid polypropylene support.

1- Concentrate secreted protein mixture

2- Prepare Immunoaffinity column

4- Elute fusion protein

3- Added secreted protein mixture to the column

Fusion protein

Other protein pass through the column

Marker peptide binds to Ab

Polypropylene supporrt

Marker peptide

Marker peptide

Interleukin-2

Anti-Marker peptide Ab

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 fragment-cloned 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.