Lecture 20 - Recombinant proteins Flashcards
Key 5 steps in producing a recombinant protein
- Isolate gene of interest
- Clone into expression plasmid –
- Tranform into bacteria for expression or isolation of more DNA for use in another
expression system - Grow cells expressing protein of interest
- Isolate and purify the protein
Advantages of prokaryotic systems
relatively low cost
high yield
pathogen free
Disadvantages of prokaryotic systems
proteins often partially folded
inability to perform post-translational modifications
Recombinant Insulin production Step 1
Reverse transcriptase
cDNA = no introns
Insulin is produced in
pancreas as pre-proprotein further processed by Golgi
– won’t happen in bacteria
Solution = express
chain A and B
separately in bacteria
Insulin Production Step 1 Step 2/3 Step 4 Step 5
Recombinant Insulin production.
Clone gene into expression plasmid(s) and transform bacteria.
Grow bacteria expressing insulin A and B chains.
Extract and purify.
Step 2-3 Clone gene into expression plasmid(s) and transform bacteria
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.
Protein purification
Recombinant insulin
Step 5. Extract and purify
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
Why Make recombinant insulin in mammalian cells?
– Protein can be produced as
a pre-pro-protein and processed efficiently
– Will be secreted from cells (easier purification)
– More expensive to produce.
Making recombinant insulin in eukaryotic cells 5 Steps
- 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
Making recombinant insulin in eukaryotic cells
Step 1.
Isolate cDNA for insulin
Making recombinant insulin in eukaryotic cells
Step 2
Clone into eukaryotic expression plasmid
Making recombinant insulin in eukaryotic cells
Step 3
Transform bacteria to produce more plasmid DNA and then transfect eukaryotic cells
Making recombinant insulin in eukaryotic cells
Step 4.
Extract recombinant insulin from cell media
Making recombinant insulin in eukaryotic cells
Step 5.
Purify insulin
Some proteins are only active when
post-translationally modified
recombinant human proteins
Therapeutic proteins –
Glycosylation
requires mammalian cells e.g. erythropoietin = EPO
EPO as a performance enhancing drug – blood doping effects
increase RBCs
increase in oxygenation of muscle
muscle uses oxygen to burn sugar and fats to generate
ATP
ATP required for muscle contraction
Why would we want to make recombinant EPO?
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
Erythropoietin (EPO) gene cloned in 1980s
protein is post-translationally modified (glycosylation)
made in Chinese Hamster Ovary (CHO) cells
glycosylation
Addition of carbohydrates to asparagine, serine or threonine residues.
important for biological function.
Pharming:
using whole animals to make recombinant proteins
Expression vector for EPO
Promoter – for transcription in mammalian cells
Human EPO
Protein purification
cells in culture cannot perform all
post-translational modifications equally well
e.g. γ carboxylation of glutamate
what is a feature of many
proteins involved in blood clotting?
γ-carboxylation of certain glutamate residues
anti-thrombin (AT)
2006 (Europe) 1st recombinant protein transgenic animal was approved as a drug
AT deficiency
hereditary or acquired
1 / 2000-5000
increase risk of blood clotting
deficient individuals receive AT when
undergoing surgery or giving birth
AT protein expressed in
milk of transgenic goats at lactation
AT then purified from other milk proteins
milk specific promoter
responds to hormonal signals that induce lactation
Recombinant DNA technologies are important for
production therapeutic proteins
provide a safe means of getting human proteins for clinical use
Eukaryotic systems must be used
for proteins that require post-translational modification for their activity
There are a variety of cell systems available for
producing recombinant proteins
