BMS3021 Lecture Notes - Lecture 2: Periplasm, Ex Vivo, Non-Homologous End Joining
30 May 2018
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Department
Course
Professor
Week 1. Protein therapy, Antibodies and therapy
and, Genetic approaches to disease therapy
PROTEIN THERAPY
• Why make proteins recombinantly?
o Ca’t get it fro edogeous sources
o For efficient and selective purification
o Quality control (large batches)
o To optimise activity/efficacy
• Heterologous hosts for protein expression
o Bacteria:
Pros
Cons
Widely used
Easy manipulation
Rapid growth
Cheap
Prokaryote – no membrane bound
organelles
Many proteins expressed are insoluble
No post-translational modifications
High endotoxin content
May have problems folding human
proteins (may not recognise and make
inclusion bodies)
o Yeast - cheapest
o Plants
o Baculovirus
o Cultured mammalian cells
o Animals
• Fusion proteins:
o Genetically fuse the gene encoding the target protein with a gene encoding a
purification tag
o When chimeric protein is expressed, the tag allows for specific capture of the fusion
protein -> allows purification of virtually any protein
Advantages
Disadvantages
Improves protein yield
Prevents proteolysis – no degradation
Facilitate protein refolding
Increase solubility
Ease of purification
Lower protein yields
-cleavage may not be complete
Alteration in biological activity
Cleavage/removing the fusion partner
may require expensive proteases
find more resources at oneclass.com
find more resources at oneclass.com
• Where to target expression of recombinant proteins in bacteria:
o Direct expression (cytosol) – do not get proper S-S bonds
o Secretion (periplasm or medium) – proteins are fused to peptides or proteins targeted
for secretion. The periplasm offers more oxidising environment, where proteins can fold
better
• Inclusion bodies in E.coli:
o Dense particles containing precipitated (insoluble) proteins
o Formation depends on protein synthesis rate and growth conditions
o Protein folding options exist but poor success rates
• Case studies:
Insulin
Erythropoietin (EPO)
o Secreted in pancreas
o Stimulated by glucose, aa and FA
o Used in type I diabetes
o Starts as single polypeptide -> into ER ->
cleave ER signal -> becomes proinsulin -> out
of ER in vesicles -> proteases in vesicle ->
create a and b chain
o Traffics through secretory pathway
o S-S bound but not glycosylated
o Proteolytically processed
History:
o Initially used animal insulin
o Patients developed immune responses due
to impurities and presence of pro-insulin
o Demands not meeting supply
o Recombinant insulin now used
-problem with E.choli – no S-S
Eukaryotic expression systems
o Yeast
o Cheap, simple genetics manipulations, has
secretory pathway, can get S-S bond
o Must have yeast specific plasmids
o Synthesised in kidney
o Stimulated RBC production in bone marrow
o Treatment for anaemia eg. kidney disease,
chemo, HIV
o Requires glycosylation for EPO action
o Requires expression in mammalian cell
culture
o Most proteins made in ER are
glycosylated~700 different mammalian
enzymes involved in different glycosylation
patterns
o Glycosylation increases half-life, helps
folding and direct traffic
Mammalian tissue culture:
o Conventional growth media = DMEM
o Need extra factors – growth hormones
o Production can be modified using serum free
media and defined growth factors – a lot
more expensive but has quality control
o Cells cultured: primary and transformed
immortal lines
- must use mammalian specific plasmids
• Protein expression in CHO cells:
o Chinese hamster ovary cells
o High density large scale fed batch cultivations are developed and scale-up technology is
well established
o Problem: low doubling time – low cell concentrations
o Very efficient secretory pathway
find more resources at oneclass.com
find more resources at oneclass.com
• Mammalian systems are more therapeutic
• Atryn – way of the future:
o Recombinant anti-thrombin alpha = anticoagulant
o Expressed under control of beta casein promotor produced in milk of goats = transgenic
expression
• Expression systems for therapeutic protein
• Top selling therapeutic proteins in 2010
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Protein therapy, antibodies and therapy and, genetic approaches to disease therapy. Protein therapy: why make proteins recombinantly, ca(cid:374)"t get it fro(cid:373) e(cid:374)doge(cid:374)ous sources, for efficient and selective purification, quality control (large batches, to optimise activity/efficacy, heterologous hosts for protein expression, bacteria: Prokaryote no membrane bound organelles: yeast - cheapest, plants, baculovirus, cultured mammalian cells, animals, fusion proteins: The periplasm offers more oxidising environment, where proteins can fold better. Inclusion bodies in e. coli: dense particles containing precipitated (insoluble) proteins, formation depends on protein synthesis rate and growth conditions, protein folding options exist but poor success rates, case studies: Eukaryotic expression systems: yeast, cheap, simple genetics manipulations, has secretory pathway, can get s-s bond, must have yeast specific plasmids, protein expression in cho cells, chinese hamster ovary cells. Igg: main serum antibody most stable, cdr loops undergo conformation change to accommodate antigen, possible mechanisms of action in therapy: Depletion - get rid of a cell completely used for lymphoma.
