Unit 3 - eukaryotic and prokaryotic drug systems

Question 1

Give some examples of recombinant proteins produced from E.coli?

Answer 1

Human Growth Hormone
Interferon alpha
Interferon gamma
Tissue plasminogen activator
Relaxin
Alpha-antitrypsin
Human insulin

Question 2

What limitations are there of bacterial expression systems?

Answer 2

Some eukaryotic proteins are unstable when expressed in bacteria, or lack biological activity
Prokaryotes lack of the ability to undertake the following post-translational modifications of cloned proteins
- glycosylation
- disulphide bond formation
Recombinant proteins expressed at high levels are stored as inclusion bodies
- need to be refolded
Prokaryotes harbour pyrogens such as LPS
- stimulate the immune system
- need to be removed from the final product

Question 3

Why is post-translational modifications important in drug production?

Answer 3

It affects the shape of the therapeutic protein
Influences biological activity
- antibodies
- enzymes
Affects protein half life in the body
- misfolded proteins are degraded by host proteases
- proteins with the wrong sugar groups such as antibodies are removed from the circulation

Question 4

What is eukaryotic post-translational modification protein phosphorylation?

Answer 4

Protein phosphorylation covalently attaches a phosphate group to an amino acid side chain or at the protein’s C or N termini
It alters the structural conformation of a protein which in the case of enzymes can result in them being activated or deactivated
The most common form of post-translational modification in eukaryotic cells with approximately 13,000 human proteins have phosphorylation sites
It plays an important role in cell signalling, gene expression and differentiation
Minor role in recombinant protein expression

Question 5

Why does post-translational modification of therapeutic proteins make disulphide bonds?

Answer 5

Disulphide bonds in proteins are formed between the thiol groups of cysteine residues
Stabilises protein structure in a number of ways
- links protein strands and helps the protein form in final structure
- forms hydrophobic regions which influence the final structure of the protein
In eukaryotic cells, disulphide bonds are generally formed in the lumen of the rough endoplasmic reticulum but not the cytosol. This is due to the oxidative environment of the endoplasmic reticulum and the reducing environment of the cytosol
Disulphide bonds are mostly found in
- secretory proteins
- lysosomal protein
- exoplasmic domains of membrane proteins

Question 6

Why are post-translational modification of therapeutic proteins glycosylated?

Answer 6

A principle co-translational and post-translation modification step in the synthesis of membrane and secreted proteins
~ 50% of all eukaryotic proteins synthesised in the rough ER are glycosylated
- enzyme directed site specific process
Two types of glycosylation exist
- O-linked oligosaccharides
- Ser, Thr linked
- linked to hydroxyl groups of the amino acids serine and threonine
- N-linked oligaccharides
- Asn linked
- linked to amide group

Question 7

Why is glycosylation important?

Answer 7

70% of protein products approved for human use as glycoproteins
They include
- antibodies
- hormones
- growth factors
- immune regulators
- interferons
Failure to glycosylate a protein with the correct combination of sugars will alter
- molecular size and charge and will result in its removal by receptors in the liver and the recticuloendothelial system
- immunogenicity
- xylose
- an oligosaccharide from plants
- pharmacological activity
- IgG heavy chain contains a single N-linked glycosylation site
- 2 sites for each IgG molecule
- aglycosylated forms display significant loss of antibody associated cell mediated cytotoxicity cannot adhere to cell surface receptors

Question 8

How does preproinsulin become proinsulin?

Answer 8

Disulphide bond formation

Question 9

How does proinsulin become insulin?

Answer 9

Trimming by carboxypeptidase B-like enzyme removes two basic residues from each of the new ends

Question 10

What is Atryn?

Answer 10

Anticoagulant antithrombin
Extracted from the milk of goats
- genetically modified to produce human antithrombin
One genetically modified goat can produce the same amount of antithrombin in a year as 90,000 blood donations

Question 11

What type of insulin was used initially?

Answer 11

Purified animal-sourced insulin was the only type of insulin available to diabetics until genetic advances occurred later with medical research

• pigs
• cows

Question 12

What are the advantages of using genetically engineered insulin?

Answer 12

Synthetic human insulin was produced in 1978 from E.coli

- the protein does not require glycosylation to be biologically active

Question 13

What are the disadvantages of using genetically engineered insulin?

Answer 13

Currently human insulin made from bacteria are almost twice as expensive as porcine insulin

Question 14

How is insulin produced in E.coli?

Answer 14

cDNA LacZ Beta-galactosidase
Clone in E.coli
Isolate product
Treat with cyanogen bromide to cleave at methionine amino acid link

Question 15

How is the Covid-19 vaccine produced?

Answer 15

Genes coding for the spike protein are identified and isolated
Genes inserted into ChAdOx1 viral vector
Injected into body
- cells express spike protein
- body produces antibodies against spoke proteins
If infected, immune system attacks SARS-CoV-2

Question 16

Why are post-translational modifications needed?

Answer 16

Affects the shape of the final protein
Influences biological activity of
- antibodies
- enzymes
Affects drug half life in the body/drug stability
- protein with the wrong sugar groups such as antibodies are removed from the circulation

Question 17

What is the function of interferon?

Answer 17

Hinder the replication of viruses
Affect the immune response
Three types
- alpha
- beta
- gamma
Used to treat multiple sclerosis
Relatively quickly broken down
- need for repeated injection

Question 18

How much less active is E.coli produced interferon?

Answer 18

10 times less

• non-glycosylated
• reduced solubility
• formation of insoluble disulphide links
• removal of sugar group

Question 19

Where are monoclonal antibodies glycosylated?

Answer 19

All antibodies are glycosylated at conserved positions in their constant regions

Question 20

Why are monoclonal antibodies glycosylated?

Answer 20

Affect antibody activity

• complement binding
• attachment to immune cells
• antibody dependent cell cytotoxicity

Question 21

What does chimerisation of monoclonal antibodies increase?

Answer 21

Persistence of the monoclonal antibody in the human circulation from 2 days (murine monoclonal antibody) to over 2 weeks (chimeric monoclonal antibody)

Question 22

What are the four types of monoclonal antibodies?

Answer 22

Animal
Chimeric
Humanized
Human

Question 23

What are the advantages of plant produced antibodies?

Answer 23

Relatively low cost to make
Potential to produce high biomass
- kg per acre
Disease free

Question 24

What plant produced antibody residues attached to the asparagine in the ER?

Answer 24

In plants and mammals core N-acetylglucosamine and mannose residues are attached to the asparagine in the ER

Question 25

What plant produced antibodies are further modified in mammals?

Answer 25

Further modification in the Golgi

• N-linked glycans
• alpha 1,6 fucose
• terminal sialic acid residues

Question 26

What plant produced antibodies are further modified in plants?

Answer 26

Modification in the Golgi

• N-linked glycans
• alpha 1,3-linked fucose
• B-1,2-linked xylose

Question 27

What is an immunoconjugate?

Answer 27

Development of the biologic class
- highly sophisticated drug delivery systems
Take advantage of the ability of the immune system’s ability to recognise very specific types of cell
Monoclonal antibodies have very high affinity and good specificity for certain cell types and are very well tolerated, especially when partially (chimeric) or fully humanised
After cell surface binding monoclonal antibodies are internalised into the cell and subject to degradation
- endocytosis

Question 28

What is an immunoconjugate also known as?

Answer 28

Antibody-Drug conjugate

Question 29

How does an antibody-drug conjugate work?

Answer 29

If the conjugate possesses suitably labile chemical bond(s), enzyme mediated bond cleavage can occur
- releases the therapeutic component within a target cell

Question 30

Why are marine organisms a surprisingly good source of anti-cancer and anti-bacterial drugs?

Answer 30

The sea is a very competitive place to live

Question 31

What are microtubules essential for?

Answer 31

Cell division and mitosis

- pull chromosomes apart in mitosis

Question 32

How can inhibiting microtubule formation prevent cancer?

Answer 32

Blocking microtubule action prevents mitosis

Question 33

What is the function of Dolastatins?

Answer 33

Bind to tubulin to prevent formation of microtubules

Question 34

What is a monoclonal antibody ? and its main structural components.

Answer 34

Monoclonal antibodies (mAb
or moAb) are monospecific
antibodies that are made by
identical immune cells that
are all clones of a unique
parent cell and as a
consequence all recognise the
same target 

Fab region: allows binding to the antigen
Fc region binds to cellular receptors and compliment
Hinge region: allows flexibility

In the shape of Y
heavy chain for bottom portion and start of Y

Question 35

What is the differences between murine, chimeric,
humanized and fully human monoclonal antibodies and
why are these differences important

Answer 35

Mouse proudced Mab
Recognised as foreign
by human immune
system
• Have murine
glycosylation patterns
• Do not bind efficiently
to immune effector cells
via FC receptors
• Removed by the liver

Mouse DNA encoding the binding
portion of a monoclonal antibody
is merged with human antibodyproducing DNA in cell culture to
yield partially mouse, partially
human monoclonal antibodies.
The cell and compliment binding
(Fc) region of the antibody has a
human glycosylation pattern 

Humanised 
Antibodies which have been
genetically engineered to
remove murine regions (T cell
epitopes) that lead to an
adverse immune response.

Fully Human
Made from Transgenic mice
which have been given human
immune system
Made using phage display

Question 36

What are main therapeutic uses of monoclonal

antibodies and in each case give examples

Answer 36

Cancer (2 marks)
• Herceptin® (trastuzumab) – inhibits cell replication
• ozogamicin (gemtuzumab) - cancer cell targeting- Monoclonal antibodies
for cancer. ADEPT, antibody directed enzyme prodrug therapy; ADCC,
antibody dependent cell-mediated cytotoxicity; CDC, complement
dependent cytotoxicity; MAb, monoclonal antibody; scFv, single-chain Fv
fragment.

Auto immune disease (2 marks)
• Omalizumab inhibits human immunoglobulin E (IgE) and is useful in
moderate-to-severe allergic asthma.
• Infliximab which are effective in rheumatoid arthritis

Infection (2 marks)
• Palivizumab (brand name Synagis which is manufactured by MedImmune)
is a humanized monoclonal antibody (IgG) to treat RSV
• Raxibacumab (GSK) – is a human monoclonal antibody (IgG) directed
against the toxin of B.anthracis.

Question 37

Why is the glycosylation status of an

antibody important.

Answer 37

Affects immunogenicity
• Causes a significant loss of antibody associated cell mediated
cytotoxicity-cannot adhere to cell surface receptors
• Affects half life in the body
• Give an example where glycosylation is important