1. Fig. 12.4
Products extracted from tissue/
primary cells
Product Extracted from....
insulin pancreas; bovine or porcine
growth hormone human pituitary glands
interferon viral activation of cells
urokinase human urine
factor VIII pooled human blood
2. Fig. 12.5
Problems of extraction from animal/
human sources
• small quantities available
• non-human proteins cause immunogenicity
• contamination with viruses or prions
- Creutzfeld-Jakob disease
- HIV from blood
4. Lecture 14 - Animal Cell Biotechnology
Animal cell products – Recombinant proteins
1. Insulin
• hormone produced by beta cells in the pancreas
→ allows glucose to pass into cells
→ suppresses excess production of sugar in the liver
and muscles
→ suppresses breakdown of fat for energy
5.
6.
7.
8. Lecture 14 - Animal Cell Biotechnology
Animal cell products – Recombinant proteins
beta cells in pancreas
preproinsulin
proinsulin
insulin + C-peptide
Butler, M. 1987. Animal cell technology: principles and products. Stony Stratford: Open University Press. P107.
9. Computer-generated image of insulin hexamers highlighting the threefold
symmetry, the zinc ion holdin it together and the histidine residues invlolved in zinc-
binding
Iinsulin
51 amino acids
5,8808 molecular weight
10. Lecture 14 - Animal Cell Biotechnology
Animal cell products – Recombinant proteins
• insulin produced from pig pancreas cells
→ structure of insulin differs slightly between species
→ the C-terminal amino acid of the B chain = alanine
(threonine in humans)
• two problems associated with porcine insulin
→ causes immunogenic response in some diabetic
patients
→ supply of pancreas fluctuates with meat trade
11. Fig. 12.6
Pig to human insulin
A (21) S- S
S S Thr
B (30) S S
B 30
Ala
13. Lecture 14 - Animal Cell Biotechnology
Animal cell products – Recombinant glycoproteins
2. Interferons
• glycoproteins that “interfere” with viral propagation in
cell cultures
• group of small proteins with 140-170 amino acids
• secretory protein produced from viral-infected
cells, induces antiviral state in neighboring cells
14. Interferon interferes with viral replication in protected cells
Butler, M. 1987. Animal cell technology: principles and products. Stony Stratford: Open University Press. P70.
15. Lecture 14 - Animal Cell Biotechnology
Animal cell products – Recombinant glycoproteins
3 main types of interferons:
1. IFN-α (25 subtypes) – produced from β -lymphocytes
2. IFN-β – fibroblasts – produced from fibroblasts
3. IFN-γ – T-lymphocytes – produced from T-lymphocytes
• mode of action not fully understood →
synthesis of host enzymes that degrade viral RNA and
inhibit protein synthesis
16. Lecture 14 - Animal Cell Biotechnology
Animal cell products – Recombinant glycoproteins
5. Erythropoietin (EPO)
• glycoprotein hormone produced
by the kidney (hypoxia triggers
EPO production)
• required for continuous red
blood cell production in bone
marrow (erythropoiesis)
• absence of EPO results in
impairment of red blood cell
production → anemia
• anemia treated with exogenous
EPO
17. Physiological role of erythropoietin
• Hematopoietic growth factor
• Produced in the kidney
• Stimulates red blood cell (erythrocyte)
maturation
• Induces homodimerization of 2 receptor
molecules
• Initiates intracellular signalling cascade
18. Therapeutic uses of EPO
Treatment of anaemia caused by :-
• chronic renal failure
• partial renal failure
• AIDS
• cancer chemotherapy
• autologous transfucion
19. Molecular characteristics of EPO
• Molecular weight: 39 kDa
• 165 amino acids
• Carbohydrate component: 35-40%
• 3 N-linked glycans to Asn at positions
24, 38, 83
• 1 O-linked glycan to Ser at position 126
21. Predicted structure of glycosylated human erythropoietin
The predicted structure of glycosylated protein human Erythropoietin . N- and O-glycans were
added to the core protein structure (pdbid 1BUY) using the Glycoprotein Builder tool at the
GLYCAM-Web site (www.glycam.com). High mannose N-linked glycans (Man9GlcNAc2) were
added at ASN 24, 38 and 83 and one O-linked glycan (a-GalNAc) at Ser126. (R.Woods)
24. Lecture 14 - Animal Cell Biotechnology
Animal cell products – Recombinant glycoproteins
carbohydrates make up ~40% (by weight) of
glycoprotein
→ important for full activity in vivo
allows EPO to remain in circulation
(removed by liver)
Egrie and Browne (2001) developed a novel form of
EPO (novel erythropoiesis-stimulating protein
(NESP))
hyper-glycosylated form of EPO with greater half-life
(3x half life of EPO)
25. Fig. 12.13
Variant glycoforms of recombinant Epo and NESP
Maximum number of sialic
acid groups in glycoform
22 (NESP)
14
12
8
O-linked glycan
N-linked glycans
26. Fig. 12.15
The biological activity of each isoform of Epo after a 30-day treatment
Increase in hematocrit from baseline 30
25
20
15
10
5
0
8 9 10 11 12 13 14
Number of sialic acid groups in Epo isoform
27. Fig. 12.14
Serum half-life of analogues of Epo with variable N-glycan sites
7
6
5
serum half-life (h)
4
3
2
1
0
rEpo 4-glycan NESP
Epo type
28. Lecture 14 - Animal Cell Biotechnology
Animal cell products – Recombinant glycoproteins
3. Plasminogen activators
• thrombosis (formation of blood clots) is a major cause
of premature death
• deposition of fibrin in the circulatory system, blocks
blood flow
• formation of insoluble fibrin controlled by clotting
cascade formed during wound healing
• t-PA (tissue-plasminogen activator) initiates fibrinolysis
(proteolytic cleavage of fibrin)
29. Therapeutic applications
• t-PA is used in diseases that feature blood
clots, - - pulmonary embolism
- myocardial infarction
- stroke
to be effective, t-PA must be administered
within the first 3 hours/ to be given
intravenously,
32. Lecture 14 - Animal Cell Biotechnology
Animal cell products – Recombinant glycoproteins
• gene for t-PA transfected into CHO-K1 cells, one of the
first recombinant products derived from mammalian
cells in 1987
→ secreted in vivo by a number of tissues
→ production stimulated by a number of
substances, including thrombin and histamine
→ half-life of t-PA varies from 2-4 min
33. Lecture 14 - Animal Cell Biotechnology
Animal cell products – Recombinant glycoproteins
4. Blood-clotting factors
• Hemophilia is a sex-linked (x-chromosome) genetic
disease
• inactive clotting cascade in blood, can’t form fibrin
→ hemophilia A – absence of factor VIII
→ hemophilia B – absence of factor IX
34. The clotting cascade
Wound surface contact
Factor XII Factor XIIa
Factor XI Factor XIa
Factor IX Factor IXa
+Factor VIII + Thrombin
Factor X Factor Xa
+Factor V
Prothrombin Thrombin
Fibrinogen Fibrin clot
35. Fig. 12.10
The clotting cascade
Wound surface contact
Factor XII Factor XIIa
Factor XI Factor XIa
Factor IX Factor IXa
+ Factor VIII + Thrombin
Factor X Factor Xa
+Factor V
Prothrombin Thrombin
Fibrinogen Fibrin clot
36. Lecture 14 - Animal Cell Biotechnology
Animal cell products – Recombinant glycoproteins
Factor VIII
• large glycoprotein (265 kDa)
• gene – 186 kB, 26 exons, 25 introns (overlapping
strands of DNA from genomic and cDNA aligned,
without introns)
• BHK cells transfected with expression vector containing
gene encoding Factor VIII
• produces biologically active protein with correct tertiary
folding and glycosylation
• stabilized by addition of Willebrand factor, normally
found as a combined protein complex in blood
37. Lecture 14 - Animal Cell Biotechnology
Animal cell products – Recombinant glycoproteins
Factor IX
• plasma glycoprotein (57 kDa) secreted by hepatocytes
• called “Christmas factor”, after first family diagnosed
with clotting deficiency
• gene cloned into rat hepatoma cell line
→ contains enzymes for post-translation modifications
38.
39. Lecture 14 - Animal Cell Biotechnology
Animal cell products – Artificial skin
• important for skin grafting (i.e. for severe burn victims)
• one method described by Hardin-Young and Parenteau
2002)
→ dermal-equivalent formed from fibroblasts
→ epidermal equivalent formed from keratinocytes
• keratinocytes and fibroblasts are derived from neonatal
foreskin tissue, lack antigen presentation
40. Fig. 12.16
The principle of gene therapy ex vivo