Therapeutic Proteins

Question 1

Bacterial Restriction Endonucleases

Answer 1

cut DNA at specific 4-8bp recognition sequence, EcoRI (palindromic) GAATTC, leaving sticky ends can be rejoined to form recombinant, insert into vector (using DNA ligase and ATP)

Question 2

Bacteria mRNA used to make cDNA

Answer 2

No introns

Question 3

Bacteria has no introns

Answer 3

Use mRNA to make cDNA and poly thymidated promoter for polyadenylated tail of mRNA

Question 4

Vectors

Answer 4

Plasmid

virus

Question 5

Control expression by

Answer 5

artificial chromosomes

Question 6

Making protein - key points (3)

Answer 6

Naturally produced in species? all machinery present
soluble? require folding
post-translational modification (N + O glycosylation only in eukaryotes)
codon in species? frequency - number tRNAs –> mistakes
product toxic?

Question 7

Advantages of using prokaryotes (5)

Answer 7

easy culture
rapid growth
expression easy to induce
easy retrieval
no ethics

Question 8

Disadvantages of using prokaryotes (5)

Answer 8

low expression
rapid degradation
no post-trans modification
can't glycosylate
doesn't secrete product

Question 9

Advantages of using simple eukaryotes

Answer 9

established tech
high product level
cost effective
easy to adapt for industry

Question 10

Pichia Pastoris

Answer 10

well defined growth and high density
simple glycosylation
expression controlled by AOX1 gene - change methanol conc - methylotropic

Question 11

saccharomyces cerevisae

Answer 11

make viral fragments for vaccine - HOV

disadvantage - addition of mannose at N-glycosylation sites

Question 12

Filamentous fungi - aspergillus

- trichoderma

Answer 12

• produce pectinases for food industry - more juice from fruit
• produce antibody fragments for medicine

Question 13

Eukaryotic algae - chlamydomonas reinhardtii

Answer 13

motile single cell - translated proteins folded and assembled

Question 14

Insects

Answer 14

express cDNA well
transcriptional processing of introns
folding and post-trans mod
protein secreted

Question 15

Mammalian cell lines advantages

Answer 15

full post-trans mod
some secrete protein
easy recovery and purification

Question 16

Mammalian cell line disadvantages

Answer 16

complex conditions
purification may be required
slow growth
expensive

Question 17

Transgenic animals

Answer 17

production of recombinant therapeutic proteins in milk

Question 18

Transgenic Plants

Answer 18

Use plant virus as vector - vaccine production
advan - easy to grow, cheap, easy drug delivery
disadvan - change taste, stable?, choice, where will gene go

Question 19

Glycosylation

Answer 19

oligosaccharides added to the protein during synthesis and processing through ER and golgi

Question 20

Glycan

Answer 20

carbohydrate structure

affects bioactivity, pharmacokinetics, secretion, in vivo clearance, solubility, recognition, antigenicity

Question 21

Main glycosidic links

Answer 21

N- –> bound to ASN residue Asn-X-Ser/Thr
Complex/Hybrid (both golgi)/High Mannose (ER)
O- –> bound to hydroxyl of Ser/Thr, hydroxylysine, hydroxyproline
short, but longer on ABO antigens

Question 22

N-linked glycan assembly

Answer 22

precursor - dolichol (lipid) linked to glycan by pyrophosphate
Oligosaccharyltransferase - enzyme attaches asn in ER
glucosidases on ER membrane
gycosidases and glycotransferases in golgi
addition sialic acid, fucose, poly-N-acetyl lactosamine

Question 23

O-linked glycan assembly

Answer 23

added post translationally to folded portein
on ser/thr in golgi
GalNAc transferase adds N-acetylygalactosamine for mucin type

Question 24

Glycosylation under control of

Answer 24

metabolism and cell growth

Question 25

Glycans Biological Activtiy

Answer 25

enhance thermal stability protection from proteolysis improve solubility inhibit aggregation

Question 26

Glycans - folding

Answer 26

carbohydrate negatively charged - interact AA side chain

Question 27

Erythropoietin

Answer 27

sialoglycoprotein - secreted by kidneys - hypoxia/low red cell mass - stimulate erythrocyte production from bone marrow

Question 28

Erythropoietin - Glycan

Answer 28

ensure adequate residence in bloodstream

Question 29

Recombinant Erythropoietin (Dongmei et al 2010)

Answer 29

Increase sialylation ---> increase half-life - not removed by asialoglycoprotein receptor

Question 30

Glycan Antibody function

Answer 30

IgG Fc has 2 N-linked complex type High fucose - effectively recruit polymorphonuclear cells low fucose - enhance NK cell activity

Question 31

Glycan Pharmacokinetics - Circulating Residence Time

Answer 31

Function of pharmacokinetic (PK) behaviour including clearance rate, serum half-life and max and min concs glycosylation affects PK

Question 32

FSH short half life

Answer 32

Glycosylation increases this Mannose and asiaglycoprotein receptors remove certain glycoforms Lenercept - terminal GlcNAc

Question 33

Growth Hormone

Answer 33

191 AA Single chain, non-glycosylated | recombinant used in HGH deficiency and wasting from AIDS

Question 34

Insulin

Answer 34

``` 51 AA (30+21) Ultra short analogue - not form hexamers/dimers so absorbed quicker Lente - intermediate Levemir - long-acting - myristyric acid attached to albumin so remains in circ longer ```

Question 35

Blood clotting proteins Haemophilia A

Answer 35

impairment of gene synthesise factor VIII - this has 25 N-glycosylation sites unglycosylated = immunogenic and cleared quickly

Question 36

Haemophilia B

Answer 36

Impair gene synthesises Factor IX - so is transfused in treatment post-trans modifications required - peptide removal,vitamin K dependent carboxylation, ser- phosporylation, tyr sulfation, glycosylation

Question 37

Interferons

Answer 37

140-170 AA - alpha (B lymphocytes) , beta (fibroblasts) and gamma (T lymphocytes) antiviral activity and retard cancer cells

Question 38

Interferon Alpha

Answer 38

Interfere cancer cells growth and mulptilication increase antigen presentation on cancer cells activate NK cells given for renal malignant/multiple myeloma + leukaemia

Question 39

Recombinant Alpha

Answer 39

treat hepatitis C - has human albumin attached, in blood for 4 weeks, no SE

Question 40

Interferon Beta (recombinant)

Answer 40

3 types - treat multiple sclerosis Betarson - non-glycosylated Avonex and rebif are glycosylated - higher specific activity and lower production of neutralising antibodies