Bacterial REs cut DNA at specific (_-_ bp) recognition sequences.
RE’s are used as a defensive measure by bacteria to cut up ____1____ genomes to stop them from ____2____.
RE's cut at specific recognition sequences, leaving what?
After RE cutting, ____1____ joins the 2 DNA strands together. This requires the ______ 2 ______ and an energy source in the form of ATP.
2. Sticky ends
Define a vector (genetics).
A carrier for genetic recombination.
A bacteriophage or plasmid which transfers genetic material into a cell, or from one bacterium to another.
We are able to use artificial chromosomes to control gene expression (turn on and off).
An example of this is the Lac operon.
Where would the Lac operon be inserted to control gene expression?
What turns it on and off?
Just before gene of interest.
The presence of the sugar lactose (on), absence of lactose (off). Also, in the presence of glucose, the lac operon will be turned off as glucose is a preferred source of energy.
Why can't E.coli and other less complex organisms make all proteins that can be formed in humans?
They are unable to make many of the post-translational modifications that human cells can.
List the advantages of recombinant expression systems in prokaryotes. (4)
- Ease of culture
- Rapid cell growth
- Expression easily induced
- Easy retrieval and purification of product
List the disadvantages of recombinant expression systems in prokaryotes. (4)
- Can have low expression levels
- Rapid and severe product degradation
- Missing post-translational modification
- Inability to glycosylate
List the advantages of recombinant expression systems in simple eukaryotes. (4)
- Established technology
- High level of product mg/g of cells
- Cost effective
- Relatively easy to adapt for industrial scale production
List simple eukaryotes used in recombinant expression systems. (7)
Methylotropic yeasts (use methanol as energy source)
- Candida boidinii
- Hansenula polymorpha
- Pichia pastoris
- Saccharomyces cerevisae
Trichoderma (filamentous fungi)
Aspergillus (filamentous fungi)
Saccharomyces cerevisiae (bakers yeast) is a simple eukaryotic expression system that has been successfully used to make viral fragments for vaccines (HPV).
However, what is the major disadvantage of using it?
The addition of mannose rich branched chains at N glycosylation sites induces an immune response (seen as foreign as human cells do not use mannose for glycosylation).
______ is used to produce pectinase which helps release juice from fruits (E.g. useful for wine industries).
Name a eukaryotic algae used as a recombinant expression system.
A viruses that infect insects.
List the advantages of recombinant expression systems in mammalian cell lines. (3)
- Perform full set of post-translational modifications
- Some cells have the ability to secrete the protein product into the culture media
- Easier recovery and purification
List the disadvantages of recombinant expression systems in mammalian cell lines. (4)
- Complex culture conditions required
- Purification from the complex culture media may be required
- Slow growth rate
Why use malign cells in expression systems?
The cells are immortal, therefore meaning they will keep producing product indefinitely.
List the advantages of recombinant expression systems in transgenic plants. (3)
- Easy to grow
- Low cost
- Easy to deliver as drug
List the disadvantages of recombinant expression systems in transgenic plants. (4)
- Does it change the taste of the food?
- How stable is it?
- Personal choice
- Where will the gene and protein get to
Therapeutic proteins have revolutionized the treatment of many previously unmet medical needs.
Despite this there are still problems with
- Low activity
- Rapid clearance
How many marketed therapeutic proteins are glycoproteins?
Half of human proteins are glycosylated with carbohydrate structures at one or more sites. Glycans are added by post-translational modification. The glycan component of a recombinant glycoprotein is very important as it affects what? (7)
- In vivo clearance
Define the quantitative aspects of glycosylation.
How much glycosylation.
Define the qualitative aspects of glycosylation.
Glycosylation occurs at which bonds, which sugars.
Glycosylation is the process whereby oligosaccharides are added to the protein during synthesis and processing through the ER and Golgi apparatus.
Glycoproteins occur as heterogeneous populations of molecules called glycoforms. The structure of a glycan is not governed by a predetermined template (as it is for proteins) and a variety of structures are possible for any one protein. The potential variability of glycoforms presents a very real difficulty to industrial production and for regulatory approval of therapeutic glycoproteins. The glycoforms produced will depend on which sugars were available at the time (bioavailability). This may depend on diet, stage of the cell cycle, any current immune response etc. (this is because these have an effect on the metabolism and therefore which sugars are produced).
Name and describe the 2 forms of glycosylation.