Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Biotechnology > Therapeutic Proteins > Flashcards

Therapeutic Proteins Flashcards

Sola and Griebenow (2010) Glycosylation of Therapeutic Proteins: An Effective Strategy to Optimize Efficacy. Biodrugs, 24, 9-21

1
Q

Bacterial Restriction Endonucleases

A

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)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Bacteria mRNA used to make cDNA

A

No introns

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Bacteria has no introns

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Vectors

A

Plasmid

virus

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Control expression by

A

artificial chromosomes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

Making protein - key points (3)

A

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?

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Advantages of using prokaryotes (5)

A 
easy culture
rapid growth
expression easy to induce
easy retrieval
no ethics
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Disadvantages of using prokaryotes (5)

A 
low expression
rapid degradation
no post-trans modification
can't glycosylate
doesn't secrete product
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Advantages of using simple eukaryotes

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Pichia Pastoris

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

saccharomyces cerevisae

A

make viral fragments for vaccine - HOV

disadvantage - addition of mannose at N-glycosylation sites

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Filamentous fungi - aspergillus

- trichoderma

A
  • produce pectinases for food industry - more juice from fruit
  • produce antibody fragments for medicine
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Eukaryotic algae - chlamydomonas reinhardtii

A

motile single cell - translated proteins folded and assembled

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Insects

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Mammalian cell lines advantages

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Mammalian cell line disadvantages

A

complex conditions
purification may be required
slow growth
expensive

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Transgenic animals

A

production of recombinant therapeutic proteins in milk

18
Q

Transgenic Plants

A

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

19
Q

Glycosylation

A

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

20
Q

Glycan

A

carbohydrate structure

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

21
Q

Main glycosidic links

A

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

22
Q

N-linked glycan assembly

A

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

23
Q

O-linked glycan assembly

A

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

24
Q

Glycosylation under control of

A

metabolism and cell growth

25
Q

Glycans Biological Activtiy

A

enhance thermal stability
protection from proteolysis
improve solubility
inhibit aggregation

26
Q

Glycans - folding

A

carbohydrate negatively charged - interact AA side chain

27
Q

Erythropoietin

A

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

28
Q

Erythropoietin - Glycan

A

ensure adequate residence in bloodstream

29
Q

Recombinant Erythropoietin (Dongmei et al 2010)

A

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

30
Q

Glycan Antibody function

A

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

31
Q

Glycan Pharmacokinetics - Circulating Residence Time

A

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

32
Q

FSH short half life

A

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

33
Q

Growth Hormone

A

191 AA Single chain, non-glycosylated

recombinant used in HGH deficiency and wasting from AIDS

34
Q

Insulin

A 
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
35
Q

Blood clotting proteins Haemophilia A

A

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

36
Q

Haemophilia B

A

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

37
Q

Interferons

A

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

38
Q

Interferon Alpha

A

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

39
Q

Recombinant Alpha

A

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

40
Q

Interferon Beta (recombinant)

A

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

Biotechnology (7 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2025 Bold Learning Solutions. Terms and Conditions