Lecture 11 recombinant protein production

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.


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


• 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


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

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)

http://www.glycam.com/

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


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,

http://en.wikipedia.org/wiki/Blood_clot

http://en.wikipedia.org/wiki/Pulmonary_embolism

http://en.wikipedia.org/wiki/Myocardial_infarction

N-glycan

disulphide bond

Fig. 12.9


Tissue-plasminogen activator

Plasminogen Plasmin

Coagulation Fibrin(insoluble)

Fibrin products(soluble)

Fibrinolysis


• 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

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


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

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

Technology

Lecture 11 recombinant protein production