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BMS3031 Molecular Mechanisms of Disease > Recombinant Proteins > Flashcards

Recombinant Proteins Flashcards

1
Q

What organism is used to make insulin?

A

Bacteria

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2
Q

What organism is used to make EPO?

A

Mammalian cells

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3
Q

What organism is used to make interferons?

A

Yeast

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4
Q

List four reasons for making recombinant proteins?

A

Lack of endogenous source
Efficient and selective purification
Quality control
Optimise activity/efficacy

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5
Q

What are the pros of using bacteria for protein expression?

A 
Widely used
Easy manipulation via plasmids
Rapid growth
Cheap
Many commercial vectors
Enhanced purification using tags
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6
Q

What are the cons of using bacteria for protein expression?

A

Many expressed proteins are insoluble
Bacteria do not have post-translational modification
High endotoxin content (can be overcome)

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7
Q

What needs to be known about a protein for a fusion protein to be made?

A

Nothing, any protein can be used.

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8
Q

What are some common tags used in fusion proteins?

A

Glutathione S-transferase (GsT)
Maltose-binding protein
His-tag (binds to nickel)

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9
Q

How can GsT be used to isolated recombinant proteins?

A

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).

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10
Q

What does a plasmid used to produce GsT-protein contain?

A

Strong promotor, GsT tag and a multiple cloning site with a protease (thrombin) and the protein.

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11
Q

What are the advantages of tags in fusion proteins?

A

Increase protein yield and stability.
Decrease proteolysis
Aid protein folding
Ease of purification.

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12
Q

What are the disadvantages of tags in fusion proteins?

A

Lower protein yields if cleavage is not complete
Alteration of biological activity
Proteases may be expensive

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13
Q

Why would you direct expressed proteins into the periplasm of the bacteria?

A

More oxidising environment aiding S-S formation.

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14
Q

What are inclusion bodies?

A

Dense particles in E. coli with aggregated proteins due to poor synthesis rate and conditions.

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15
Q

Which proteins are most likely to form inclusion bodies?

A

Mammalian proteins

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16
Q

When may inclusion bodies be beneficial?

A

If they can be extracted and refolded into proteins.

17
Q

Which cells secrete insulin?

A

Beta cells of isles of Langerhans in the pancreas.

18
Q

What is insulin used to treat?

A

Type I diabetes

19
Q

What fusion protein is used to make recombinant insulin in bacteria?

A

Beta-galactocydase

20
Q

How can insulin be made in bacteria if bacteria cytosol does not have S-S formation capacity?

A

Synthesise A and B chains separately as fusion proteins in bacteria using Beta galactocydase, purify and oxidise.

21
Q

How can insulin be made in yeast if it lacks the enzymes needed to remove the C chain?

A

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.

22
Q

Why does EPO have to be synthesised in mammalian cells?

A

3 glycosylated sites required for functioning

23
Q

Where and how does glycosylation of most proteins occur?

A

ER, addition of a common N-linked oligosaccharide.

24
Q

What is the purpose of glycosylation?

A

Trafficking, folding, stability, function.

25
Q

What is used to culture mammalian cells?

A

Dulbecco’s modified Eagle’s media with added foetal calf serum.

26
Q

Give an example of a strong promotor.

A

Viral, e.g. CMV (cytomegalovirus).

27
Q

What was the first therapeutic protein to be made is CHO cells?

A

Tissue plasminogen activator (tPA).

28
Q

What are the cell growth times for bacteria, yeast and mammals?

A

30 minutes, 90 minutes, 24 hours, respectively.

29
Q

What is expression like in bacteria, yeast and mammals?

A

High, medium-high, low-medium, respectively.

30
Q

What is Atryn?

A

Recombinant anti-thrombin alpha, anti-coagulant.

31
Q

How can Atryn be made?

A

Modify goat DNA to produce antithrombin, inject into goat zygote and implant. Assess born goats and breed promising ones.

BMS3031 Molecular Mechanisms of Disease (21 decks)

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