Lecture 20 - Recombinant proteins

Question 1

Key 5 steps in producing a recombinant protein

Answer 1

1. Isolate gene of interest
2. Clone into expression plasmid –
3. Tranform into bacteria for expression or isolation of more DNA for use in another
   expression system
4. Grow cells expressing protein of interest
5. Isolate and purify the protein

Question 2

Advantages of prokaryotic systems

Answer 2

relatively low cost
high yield
pathogen free

Question 3

Disadvantages of prokaryotic systems

Answer 3

proteins often partially folded

inability to perform post-translational modifications

Question 4

Recombinant Insulin production Step 1

Answer 4

Reverse transcriptase

cDNA = no introns

Question 5

Insulin is produced in

Answer 5

pancreas as pre-proprotein further processed by Golgi
– won’t happen in bacteria

Solution = express
chain A and B
separately in bacteria

Question 6

Insulin Production
Step 1
Step 2/3
Step 4
Step 5

Answer 6

Recombinant Insulin production.
Clone gene into expression plasmid(s) and transform bacteria.
Grow bacteria expressing insulin A and B chains.
Extract and purify.

Question 7

Step 2-3 Clone gene into expression plasmid(s) and transform bacteria

Answer 7

lac Z gene
Promoter
Antibiotic resistance gene
Insulin Gene A or B subunit

Transforms in E. Coli host

lac Z / Insulin A or B fusion protein accumulates in cell.

Question 8

Protein purification

Answer 8

Recombinant insulin

Question 9

Step 5. Extract and purify

Answer 9

lac Z / insulin fusion proteins

Treat with Cyanogen bromide to cleave A and B chains

Purify mix A and B chains to form functional insulin

Question 10

Why Make recombinant insulin in mammalian cells?

Answer 10

– Protein can be produced as
a pre-pro-protein and processed efficiently

– Will be secreted from cells (easier purification)

– More expensive to produce.

Question 11

Making recombinant insulin in eukaryotic cells 5 Steps

Answer 11

• Step 1. Isolate cDNA for insulin
• Step 2. Clone into eukaryotic expression plasmid
• Step 3. Transform bacteria to produce more plasmid DNA and then transfect eukaryotic cells
• Step 4. Extract recombinant insulin from cell media
• Step 5. Purify insulin

Question 12

Making recombinant insulin in eukaryotic cells

Step 1.

Answer 12

Isolate cDNA for insulin

Question 13

Making recombinant insulin in eukaryotic cells

Step 2

Answer 13

Clone into eukaryotic expression plasmid

Question 14

Making recombinant insulin in eukaryotic cells

Step 3

Answer 14

Transform bacteria to produce more plasmid DNA and then transfect eukaryotic cells

Question 15

Making recombinant insulin in eukaryotic cells

Step 4.

Answer 15

Extract recombinant insulin from cell media

Question 16

Making recombinant insulin in eukaryotic cells

Step 5.

Answer 16

Purify insulin

Question 17

Some proteins are only active when

Answer 17

post-translationally modified

Question 18

recombinant human proteins

Answer 18

Therapeutic proteins –

Question 19

Glycosylation

Answer 19

requires mammalian cells e.g. erythropoietin = EPO

Question 20

EPO as a performance enhancing drug – blood doping effects

Answer 20

increase RBCs
increase in oxygenation of muscle

muscle uses oxygen to burn sugar and fats to generate
ATP

ATP required for muscle contraction

Question 21

Why would we want to make recombinant EPO?

Answer 21

restore RBC levels

lowered RBC counts.

chronic renal failure decrease in EPO levels, leading to anaemia (decrease
in RBC levels)

cancer treatments (chemotherapy) may lead to anaemia

Question 22

Erythropoietin (EPO) gene cloned in 1980s

Answer 22

protein is post-translationally modified (glycosylation)

made in Chinese Hamster Ovary (CHO) cells

Question 23

glycosylation

Answer 23

Addition of carbohydrates to asparagine, serine or threonine residues.

important for biological function.

Question 24

Pharming:

Answer 24

using whole animals to make recombinant proteins

Question 25

Expression vector for EPO

Answer 25

Promoter – for transcription in mammalian cells

Human EPO

Protein purification

Question 26

cells in culture cannot perform all

Answer 26

post-translational modifications equally well

e.g. γ carboxylation of glutamate

Question 27

what is a feature of many

proteins involved in blood clotting?

Answer 27

γ-carboxylation of certain glutamate residues

Question 28

anti-thrombin (AT)

Answer 28

2006 (Europe) 1st recombinant protein transgenic animal was approved as a drug

Question 29

AT deficiency

Answer 29

hereditary or acquired

1 / 2000-5000

increase risk of blood clotting

Question 30

deficient individuals receive AT when

Answer 30

undergoing surgery or giving birth

Question 31

AT protein expressed in

Answer 31

milk of transgenic goats at lactation

AT then purified from other milk proteins

Question 32

milk specific promoter

Answer 32

responds to hormonal signals that induce lactation

Question 33

Recombinant DNA technologies are important for

Answer 33

production therapeutic proteins

provide a safe means of getting human proteins for clinical use

Question 34

Eukaryotic systems must be used

Answer 34

for proteins that require post-translational modification for their activity

Question 35

There are a variety of cell systems available for

Answer 35

producing recombinant proteins