lecture 11 recombinant protein production
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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
Fig. 12.4
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
Fig. 12.5
Fig. 12.17
Lecture 14 - Animal Cell BiotechnologyAnimal 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
beta cells in pancreas
preproinsulin
proinsulin
insulin + C-peptide
Butler, M. 1987. Animal cell technology: principles and products. Stony Stratford: Open University Press. P107.
Lecture 14 - Animal Cell BiotechnologyAnimal cell products – Recombinant proteins
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 acids5,8808 molecular weight
Lecture 14 - Animal Cell BiotechnologyAnimal 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
Pig to human insulin
S- S
SS
SS
A (21)
B (30)Thr
Ala
B 30
Fig. 12.6
Producing A and B chains separately
Lecture 14 - Animal Cell BiotechnologyAnimal 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
Butler, M. 1987. Animal cell technology: principles and products. Stony Stratford: Open University Press. P70.
Interferon interferes with viral replication in protected cells
Lecture 14 - Animal Cell BiotechnologyAnimal 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
Lecture 14 - Animal Cell BiotechnologyAnimal 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
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
Therapeutic uses of EPO
Treatment of anaemia caused by :-• chronic renal failure• partial renal failure• AIDS• cancer chemotherapy• autologous transfucion
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
Structure of erythropoietinFig. 12.11
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)
Recombinant human Erythropoietin
Ser126
Asn24
Asn38Asn83
Non-glycosylated Glycosylated
18 kDa
39 kDa
Fig. 12.12
= Fuc
= GlcNAc
= Man
= Gal
= NeuAc
Asn-X-Ser/Thr
Tetra-antennary N-glycan structure
Asn
Complextetra-antennary
a2-3
b1-2
a1-6 a1-6
b1-4
a1-3
-linkage
-linkage
23
4
86
Linkage position
Lecture 14 - Animal Cell BiotechnologyAnimal 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)
Maximum number of sialic acid groups in glycoform
22 (NESP)
14
12
8
N-linked glycansO-linked glycan
Variant glycoforms of recombinant Epo and NESPFig. 12.13
The biological activity of each isoform of Epo after a 30-day treatment
Number of sialic acid groups in Epo isoform
8 9 10 11 12 13 14
Incr
ea
se in
he
ma
tocr
it fr
om
ba
selin
e
0
5
10
15
20
25
30
Fig. 12.15
Serum half-life of analogues of Epo with variable N-glycan sites
Epo type
rEpo 4-glycan NESP
seru
m h
alf-
life
(h
)
0
1
2
3
4
5
6
7
Fig. 12.14
Lecture 14 - Animal Cell BiotechnologyAnimal 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)
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,
N-glycan
disulphide bond
Fig. 12.9
Lecture 14 - Animal Cell BiotechnologyAnimal cell products – Recombinant glycoproteins
Tissue-plasminogen activator
Plasminogen Plasmin
Coagulation Fibrin(insoluble)
Fibrin products(soluble)
Fibrinolysis
Lecture 14 - Animal Cell BiotechnologyAnimal 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
Lecture 14 - Animal Cell BiotechnologyAnimal 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
Wound surface contact
Factor XII Factor XIIa
Factor XI Factor XIa
Factor IX Factor IXa
Factor X Factor Xa
Prothrombin Thrombin
Fibrinogen Fibrin clot
+Factor VIII + Thrombin
+Factor V
The clotting cascade
The clotting cascade
Wound surface contact
Factor XII Factor XIIa
Factor XI Factor XIa
Factor IX Factor IXa
+ Factor VIII +
Factor X Factor Xa
Prothrombin Thrombin
Fibrinogen Fibrin clot
+Factor V
Thrombin
Fig. 12.10
Lecture 14 - Animal Cell BiotechnologyAnimal 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
Lecture 14 - Animal Cell BiotechnologyAnimal 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
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
The principle of gene therapy ex vivoFig. 12.16
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