Recombinant Proteins Flashcards
What organism is used to make insulin?
Bacteria
What organism is used to make EPO?
Mammalian cells
What organism is used to make interferons?
Yeast
List four reasons for making recombinant proteins?
Lack of endogenous source
Efficient and selective purification
Quality control
Optimise activity/efficacy
What are the pros of using bacteria for protein expression?
Widely used Easy manipulation via plasmids Rapid growth Cheap Many commercial vectors Enhanced purification using tags
What are the cons of using bacteria for protein expression?
Many expressed proteins are insoluble
Bacteria do not have post-translational modification
High endotoxin content (can be overcome)
What needs to be known about a protein for a fusion protein to be made?
Nothing, any protein can be used.
What are some common tags used in fusion proteins?
Glutathione S-transferase (GsT)
Maltose-binding protein
His-tag (binds to nickel)
How can GsT be used to isolated recombinant proteins?
Poor through a column with glutathione to isolate GsT-protein.
Poor glutathione through column to remove GsT-protein from column.
Cleave tag off using protease (thrombin).
What does a plasmid used to produce GsT-protein contain?
Strong promotor, GsT tag and a multiple cloning site with a protease (thrombin) and the protein.
What are the advantages of tags in fusion proteins?
Increase protein yield and stability.
Decrease proteolysis
Aid protein folding
Ease of purification.
What are the disadvantages of tags in fusion proteins?
Lower protein yields if cleavage is not complete
Alteration of biological activity
Proteases may be expensive
Why would you direct expressed proteins into the periplasm of the bacteria?
More oxidising environment aiding S-S formation.
What are inclusion bodies?
Dense particles in E. coli with aggregated proteins due to poor synthesis rate and conditions.
Which proteins are most likely to form inclusion bodies?
Mammalian proteins
When may inclusion bodies be beneficial?
If they can be extracted and refolded into proteins.
Which cells secrete insulin?
Beta cells of isles of Langerhans in the pancreas.
What is insulin used to treat?
Type I diabetes
What fusion protein is used to make recombinant insulin in bacteria?
Beta-galactocydase
How can insulin be made in bacteria if bacteria cytosol does not have S-S formation capacity?
Synthesise A and B chains separately as fusion proteins in bacteria using Beta galactocydase, purify and oxidise.
How can insulin be made in yeast if it lacks the enzymes needed to remove the C chain?
Replace C chain with Arg and Lys and insert a yeast ER sequence. Once the peptide is synthesised, used trypsin to cleave Arg and Carboxypeptidase to cleave lysine.
Why does EPO have to be synthesised in mammalian cells?
3 glycosylated sites required for functioning
Where and how does glycosylation of most proteins occur?
ER, addition of a common N-linked oligosaccharide.
What is the purpose of glycosylation?
Trafficking, folding, stability, function.
What is used to culture mammalian cells?
Dulbecco’s modified Eagle’s media with added foetal calf serum.
Give an example of a strong promotor.
Viral, e.g. CMV (cytomegalovirus).
What was the first therapeutic protein to be made is CHO cells?
Tissue plasminogen activator (tPA).
What are the cell growth times for bacteria, yeast and mammals?
30 minutes, 90 minutes, 24 hours, respectively.
What is expression like in bacteria, yeast and mammals?
High, medium-high, low-medium, respectively.
What is Atryn?
Recombinant anti-thrombin alpha, anti-coagulant.
How can Atryn be made?
Modify goat DNA to produce antithrombin, inject into goat zygote and implant. Assess born goats and breed promising ones.
