Lecture 11 recombinant protein production

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

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

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


beta cells in pancreas

preproinsulin

proinsulin

insulin + C-peptide

Butler, M. 1987. Animal cell technology: principles and products. Stony Stratford: Open University Press. P107.

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


• 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

Fig. 12.6

Pig to human insulin

A (21) S- S

S S Thr
B (30) S S

B 30

Ala

Producing A and B chains separately

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

Interferon interferes with viral replication in protected cells

Butler, M. 1987. Animal cell technology: principles and products. Stony Stratford: Open University Press. P70.


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

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

Fig. 12.11
Structure of erythropoietin

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)

Fig. 12.12 Recombinant human Erythropoietin

Non-glycosylated Glycosylated

Asn83
Asn38

Asn24

Ser126

39 kDa

18 kDa

Tetra-antennary N-glycan structure

1-4
Asn-X-Ser/Thr
1-6 1-6
= Fuc
Asn
= GlcNAc
1-3
8
= Man 6
1-2
4
= Gal
3
2
= NeuAc 2-3 Linkage position

Complex -linkage
-linkage
tetra-antennary

 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)

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

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

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


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,

Fig. 12.9

disulphide bond

N-glycan

Fibrinolysis
Tissue-plasminogen activator

Plasminogen Plasmin

Coagulation Fibrin Fibrin products
(insoluble) (soluble)


• 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


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

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

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

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


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

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

Fig. 12.16
The principle of gene therapy ex vivo

Lecture 11 recombinant protein production

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Viewers also liked

Viewers also liked (20)

Similar to Lecture 11 recombinant protein production

Similar to Lecture 11 recombinant protein production (20)

More from Sarah Aira Santos

More from Sarah Aira Santos (15)

Recently uploaded

Recently uploaded (20)

Lecture 11 recombinant protein production