Expression systems Flashcards
Cloned genes can be expressed in a variety of systems. The most common systems are:
- E. coli
- Yeast
- Mammalian
Post translational modification examples include.
Some proteins need to be modified further after they’ve been translated such as:
- cleavage of precursor proteins (insulin)
- correct folding (H bonds, disulphide bonds)
- amino acid modifications (acylation, methylation, phosphorylation)
- glycosylation (addition of carbohydrate chains to form glycoproteins).
Glycosylation can modify:
- protein folding
- cellular localisation
- protein activity
What are some therapeutic proteins that are glycosylated when produced naturally in the body? How does the level of glycosylation affect these proteins?
- most interleukins and interferons
- CSF
- TNF
- gonadotrophins
- blood factors
- EPO
- TPA
- Monoclonal antibodies
- Lack of glycosylation can be damaging to the function of some but not all of these proteins.
Why do we use E.coli as a host cell system? What are the advantages and disadvantages?
They are cheap and produce lots of proteins and it is the easiest organism to genetically manipulate.
PROBLEM: E.coli cannot make all of the proteins of interest.
What type of cells does glycosylation occur in? As a result, which of these can make glycoproteins?
Eukaryote and yeast cells NOT prokaryotic cells (E.coli), therefore, glycoproteins need to be made in eukaryotic expression systems.
Compare and contrast the general features of proteins produced in different systems (e.coli, yeast and mammalian.
- Concentration of protein: E.coli (high; 30%), yeast (high; 5%), mammalian cells (low)
- Molecular weight: E.coli (low), yeast (high), mammalian cells (high)
- Folding: E.coli (misfolding), yeast (correct folding), mammalian cells (correct folding)
- Glycosylation: E.coli (no), yeast (yes), mammalian cells (yes)
- Retrovirus: E.coli (no), yeast (no), mammalian cells (yes)
- Pyrogen: E.coli (yes), yeast (no), mammalian cells (no)
Component of expression systems are:
- expression host (cells)
- a vector system (plasmid)
- a means of introducing vectors into the host (chemical transformation, electrotransformation or lipofection)
Examples of proteins made in E.coli?
- Insulin
- Tumour necrosis factor
- IL-2
- Interferon alpha, beta and gamma
- Human growth hormon
What are some advantages of using yeast as a expression host?
- Well characterised, fast growing, cheap, safe
- does post translational modification
- secretes a few proteins normally
What are some disadvantages of using yeast as a expression host?
- expression levels is
What are some proteins made in yeast cells?
- Engineered short acting insulin
- Fibroblast growth factor
- Hirudin anticoagulant
- G-MCSF
- Hep-B, malarial and other antigens
Give some examples of mammalian expression systems:
HEK, Human embryonic kidney cells
Chinese Hamster Ovary cells, CHO
Baby Hamster Kidney cells, BHK
Advantages of mammalian expression systems:
- post translational modification
- correct folding
Disadvantages of mammalian expression systems:
- slow growing, expensive
- risk of viral contamination from cells, growth media or viral vectors
