Expression systems

Question 1

Cloned genes can be expressed in a variety of systems. The most common systems are:

Answer 1

1. E. coli
2. Yeast
3. Mammalian

Question 2

Post translational modification examples include.

Answer 2

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).

Question 3

Glycosylation can modify:

Answer 3

1. protein folding
2. cellular localisation
3. protein activity

Question 4

What are some therapeutic proteins that are glycosylated when produced naturally in the body? How does the level of glycosylation affect these proteins?

Answer 4

• 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.

Question 5

Why do we use E.coli as a host cell system? What are the advantages and disadvantages?

Answer 5

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.

Question 6

What type of cells does glycosylation occur in? As a result, which of these can make glycoproteins?

Answer 6

Eukaryote and yeast cells NOT prokaryotic cells (E.coli), therefore, glycoproteins need to be made in eukaryotic expression systems.

Question 7

Compare and contrast the general features of proteins produced in different systems (e.coli, yeast and mammalian.

Answer 7

• 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)

Question 8

Component of expression systems are:

Answer 8

1. expression host (cells)
2. a vector system (plasmid)
3. a means of introducing vectors into the host (chemical transformation, electrotransformation or lipofection)

Question 9

Examples of proteins made in E.coli?

Answer 9

• Insulin
• Tumour necrosis factor
• IL-2
• Interferon alpha, beta and gamma
• Human growth hormon

Question 10

What are some advantages of using yeast as a expression host?

Answer 10

• Well characterised, fast growing, cheap, safe
• does post translational modification
• secretes a few proteins normally

Question 11

What are some disadvantages of using yeast as a expression host?

Answer 11

• expression levels is

Question 12

What are some proteins made in yeast cells?

Answer 12

• Engineered short acting insulin
• Fibroblast growth factor
• Hirudin anticoagulant
• G-MCSF
• Hep-B, malarial and other antigens

Question 13

Give some examples of mammalian expression systems:

Answer 13

HEK, Human embryonic kidney cells
Chinese Hamster Ovary cells, CHO
Baby Hamster Kidney cells, BHK

Question 14

Advantages of mammalian expression systems:

Answer 14

• post translational modification

- correct folding

Question 15

Disadvantages of mammalian expression systems:

Answer 15

• slow growing, expensive

- risk of viral contamination from cells, growth media or viral vectors

Question 16

In a retroviral VECTOR, all components required for making live virus particles are removed. These are:
What parts of the rectroviral vector are RETAINED?

Answer 16

gag, pro, pol, env

LTR (long terminal repeats), packaging signal and sites of insertion into the host genome

Question 17

LTR (Long terminal repeats) serve as

Answer 17

the promoter and terminator of the gene

Question 18

READ:

Answer 18

Once the gene is cloned it is used to transfect a packaging cell line containing the gag, pol and env genes.
The recombinant retrovirus is produced.
Recombinant virus can now be used to infect a host.
The RNA genome is reverse transcribed and inserted into genome.
The gene of interest is expressed.

Question 19

What are some proteins made in mammalian cells?

Answer 19

Monoclonal antibodies
Interleukin 2
Interferon beta and gamma
Growth hormone