P1252109393TIhdE

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Overexpression of mammalian integral membrane
proteins Chris Tate MRC Laboratory of
Molecular Biology Cambridge
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Why do you want to express a membrane protein?
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Why do you want to express a membrane protein?
Structural studies
Electrophysiology
1 -10 million copies/cell (1-10 mg/L)
1000-100,000 copies/cell (depending on
conductance)
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Why do you want to express a membrane protein?
Structural studies
Electrophysiology
1 -10 million copies/cell (1-10 mg/L)
1000-100,000 copies/cell (depending on
conductance)
Does not matter whether the protein is in the ER
or plasma membrane
Must be on the plasma membrane
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Why do you want to express a membrane protein?
Structural studies
Electrophysiology
1 -10 million copies/cell (1-10 mg/L)
1000-100,000 copies/cell (depending on
conductance)
Does not matter whether the protein is in the ER
or plasma membrane
Must be on the plasma membrane
Expression system should be easy to scale up
Small-scale culture sufficient
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Why do you want to express a membrane protein?
Structural studies
Electrophysiology
1 -10 million copies/cell (1-10 mg/L)
1000-100,000 copies/cell (depending on
conductance)
Does not matter whether the protein is in the ER
or plasma membrane
Must be on the plasma membrane
Expression system should be easy to scale up
Small-scale culture sufficient
All expressed protein throughout the cell should
be fully functional
Plasma membrane localised protein must be fully
functional
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Why do you want to express a membrane protein?
Structural studies
Electrophysiology
1 -10 million copies/cell (1-10 mg/L)
1000-100,000 copies/cell (depending on
conductance)
Does not matter whether the protein is in the ER
or plasma membrane
Must be on the plasma membrane
Expression system should be easy to scale up
Small-scale culture sufficient
All expressed protein throughout the cell should
be fully functional
Plasma membrane localised protein must be fully
functional
Homogeneous post-translational modifications
Post-translational modifications may be important
depending upon the experiment
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Structures of mammalian polytopic membrane
proteins
Cytochrome c oxidase 1996 Bovine heart
mitochondria Cytochrome bc1 complex 1997
Bovine heart mitochondria Ca2-ATPase 2000 Rabb
it sarcoplasmic reticulum Rhodopsin 2000 Bovine
rod outer segments Aquaporin 1 2000 Human red
blood cells ADP/ATP carrier 2003 Bovine heart
mitochondria Aquaporin 0 2004 Bovine eye lens
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Structures of mammalian polytopic membrane
proteins
Cytochrome c oxidase 1996 Bovine heart
mitochondria Cytochrome bc1 complex 1997
Bovine heart mitochondria Ca2-ATPase 2000 Rabb
it sarcoplasmic reticulum Rhodopsin 2000 Bovine
rod outer segments Aquaporin 1 2000 Human red
blood cells ADP/ATP carrier 2003 Bovine heart
mitochondria Aquaporin 0 2004 Bovine eye lens
High natural abundance Very stable in membranes
and detergent (except AAC1) No structures
obtained from over-expressed polytopic
mammalian membrane proteins
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Choice of expression system What are the options?
In vitro synthesis - chemical synthesis
- transcription/translation systems Archaebact
eria (Halobacterium salinarum) Eubacteria - Gram
negative (Escherichia coli) - Gram positive
(Lactococcus lactis) Yeasts (Saccharomyces
cerevisiae, Pichia pastoris) Insect cells - the
baculovirus expression system - Schneider (S2)
stable cell lines Mammalian cells - stable cell
lines (constitutive or inducible) -
alphaviruses (Sindbis or Semliki Forest)
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Choice of expression system Which do I choose?
E. coli
Yeast
Baculovirus
Mammalian cells
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Choice of expression system Which do I choose?
Types of proteins
Advantages disadvantages
All bacterial membrane proteins Some mammalian
membrane proteins e.g. G protein coupled
receptors Soluble domains of membrane proteins
Simple, fast, cheap, effective Few
post-translational
    modifications
E. coli
Yeast
Baculovirus
Mammalian cells
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Choice of expression system Which do I choose?
Types of proteins
Advantages disadvantages
Simple, fast, cheap, effective Few
post-translational
    modifications
All bacterial membrane proteins Some mammalian
membrane proteins e.g. G protein coupled
receptors Soluble domains of membrane proteins
E. coli
Useful for many mammalian membrane proteins e.g.
ABC transporters, GPCRs. Excellent for plant
channels and transporters
Simple cheap Post-translational
modifications Proteolysis can be a problem
Yeast
Baculovirus
Mammalian cells
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Choice of expression system Which do I choose?
Types of proteins
Advantages disadvantages
All bacterial proteins Many mammalian soluble
proteins and domains Some mammalian membrane
proteins
Simple, fast, cheap, effective Few
post-translational
    modifications
E. coli
Useful for many mammalian membrane proteins e.g.
ABC transporters, GPCRs. Excellent for plant
channels and transporters
Simple cheap Post-translational
modifications Proteolysis can be a problem
Yeast
Time consuming expensive All mammalian
post-    translational modifications Misfolded
protein can occur
Most mammalian membrane proteins Proteins with
N-glycans,    palmitoylation, phosphorylation
Baculovirus
Mammalian cells
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Choice of expression system Which do I choose?
Types of proteins
Advantages disadvantages
All bacterial proteins Many mammalian soluble
proteins and domains Some mammalian membrane
proteins
Simple, fast, cheap, effective Few
post-translational
    modifications
E. coli
Useful for many mammalian membrane proteins e.g.
ABC transporters, GPCRs. Excellent for plant
channels and transporters
Simple cheap Post-translational
modifications Proteolysis can be a problem
Yeast
Time consuming expensive All mammalian
post-    translational modifications Misfolded
protein can occur
Most mammalian membrane proteins Proteins with
N-glycans,    palmitoylation, phosphorylation
Baculovirus
Protein all likely to be functional Time
consuming expensive Often low levels of
expression
Mammalian cells
Complex mammalian proteins
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Serotonin transporter (SERT) Predictions from the
cDNA sequence
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Drugs and antidepressants that interact with the
serotonin transporter
SEROTONIN
AMPHETAMINE
CH2
NH2
CH
N
MDMA (ECSTASY)
CH3
OH
CH2
CH2
SUBSTRATE
ABUSED DRUGS
NH3

COCAINE
IMIPRAMINE
ANTIDEPRESSANTS
NCH3
O
C
FLUOXETINE
(PROZAC)
O
INHIBITOR
125
I-RTI55
NCH3
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The seven expression systems used for the
production of SERT
1. Escherichia coli 2. Pichia pastoris 3.
Baculovirus 4. Mammalian cell lines (a)
constitutive expression 293EBNA system (b)
inducible expression (i) MEL cells DMSO
inducible (ii) pCytTS cold inducible (iii)
T-Rex tetracycline inducible
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Escherichia coli
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Western blot of SERT expressed in Escherichia coli
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Pichia pastoris
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Western blot of SERT expressed in Pichia pastoris
UI uninduced I induced H endoH
treatment F PNGaseF treatment HM
high-mannose N-glycan UG unglycosylated
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N.B.
Simpler mammalian membrane proteins can be
functionally expressed at high levels in
bacteria, yeast and using an in vitro
transcription/translation system in the presence
of detergent
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G protein-coupled receptors (GPCRs)
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Expression of the 5HT5A and b2 adrenergic
receptors in Pichia pastoris
Weiss et al (1998) Biochem. J. 330, 1137-1147
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Functional expression of the neurotensin receptor
in E. coli
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Non-functional expression of the leukotriene B4
receptor in E. coli
Baneres et al., (2003) J. Mol. Biol. 329, 801-814
cDNA re-synthseized with optimal E. coli codon
usage
Expressed using T7 promoter at 37C - inclusion
bodies
BLT1 purified in 6M urea by Ni-NTA column 10
mg/L cells
Purified BLT1 refolded on NiNTA column by
applying a linear gradient of urea (6 - 0 M) in
the presence of detergent
Aggregates removed by ultracentrifugation
Final yield of 2 - 3 mg/L of cells
BLT1 bound agonist with high-affinity with n1
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Functional expression of GPCRs in a cell-free
translation system
Ishihara et al (2005) Protein Exp. Purif. 41,
27-37
N-terminal thioredoxin (no signal peptide)
C-terminal His6 tag for purification, or G protein
Cell-free protein expression with an E. coli S30
extract
Two detergents (digitonin or Brij35) kept the
GPCRs soluble
Up to 1 mg/ml produced in 6-8 hours
Expressed TRX-b2AR-Gsa bound antagonist with high
affinity (5.5 1.1 nM) although agonist binding
was weaker than expected
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Baculovirus expression system
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5-HT uptake into cells
Expression of SERT in insect cells
Western blot
125I-RTI55 binding
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N-glycosylation improves the efficiency of SERT
folding and is not required for 5HT transport or
inhibitor binding
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Western blot of SERT expressed in various insect
cell lines
Lanes 1-4 contain an identical amount of
functional SERT (125I-RTI55 binding)
1 2 3 4 tun
Conclusion The majority of unglycosylated SERT
and the higher molecular weight form is inactive
1. Sf9 2. Sf21 3. Hi5 4. MG1 tun tunicamycin
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SERT expression in insect cells using the
baculovirus expression system
Functional expression levels 250,000 copies per
cell (0.03 mg/L)
Uptake of 3H-serotonin into cells very low,
implying that only a tiny proportion of SERT is
in the plasma membrane
Non-functional SERT abundant
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Improving the functional expression of SERT
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Improving the functional expression of SERT
At which steps during the synthesis of SERT is it
possible to intervene and improve functional
expression?
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Transporters, ion channels and G protein coupled
receptors which have been shown to interact with
molecular chaperones
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Co-expression of molecular chaperones reduces the
production of misfolded SERT
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Why is SERT expressed in a functional form in the
baculovirus expression system, but it is inactive
when produced in Pichia pastoris or Escherichia
coli?
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Why is SERT expressed in a functional form in the
baculovirus expression system, but it is inactive
when produced in Pichia pastoris or Escherichia
coli?
CHOLESTEROL ?
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Specific 3H-imipramine binding (percentage)
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Specific 3H-imipramine binding (percentage)
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Factors that affect serotonin transporter
expression
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Mammalian cell expression systems
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Mammalian cell expression systems
1. Stable cell line, constitutive expression a.
integration into genome b. episomal plasmid
EBNA293 system 2. Stable cell line, inducible
expression (integration into genome) a. MEL cell
system b. pCytTS system c. T-Rex system 3.
Transient expression e.g. Semliki Forest Virus
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EBNA293 system Invitrogen
The EBV origin of replication (oriP) and nuclear
antigen (EBNA-1) allows episomal
replication. Constitutive expression from the
RSV promoter
1. Transfect HEK293 cells and select for
hygromycin-resistance 2. Inhibit SERT by
addition of cocaine or imipramine to the cell
culture medium
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MEL cell system (AstraZeneca)
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pCytTS system Cytos
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The pCytTS expression system Cytos
Biotechnology AG
Replicase nsp1-4
SERT cDNA
PSG
PRSV
DNA
5 NTR
3 NTR
TRANSCRIPTION
mRNA
()
AAAn
TRANSLATION
37C
INACTIVE
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The pCytTS expression system Cytos
Biotechnology AG
Replicase nsp1-4
SERT cDNA
PSG
PRSV
DNA
5 NTR
3 NTR
TRANSCRIPTION
mRNA
()
AAAn
29-35C
REPLICATION
TRANSLATION
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The pCytTS expression system Cytos
Biotechnology AG
Replicase nsp1-4
SERT cDNA
PSG
PRSV
DNA
5 NTR
3 NTR
TRANSCRIPTION
mRNA
()
AAAn
29-35C
REPLICATION
TRANSLATION
REPLICATION
mRNA
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The pCytTS expression system Cytos
Biotechnology AG
Replicase nsp1-4
SERT cDNA
PSG
PRSV
DNA
5 NTR
3 NTR
TRANSCRIPTION
mRNA
()
AAAn
29-35C
REPLICATION
TRANSLATION
REPLICATION
mRNA
TRANSLATION
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T-Rex system Invitrogen
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Results
1. Inhibitor binding 2. Serotonin uptake 3.
Intracellular localisation 4. Western blots
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Comparison of functional SERT expression levels
as measured by 125I-RTI55 binding
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Uptake of 3H-5HT into mammalian cells
expressing SERT
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Summary of 3H-5HT uptake data for different
mammalian cell lines

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Confocal images of immunostained cells expressing
SERT
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Comparison of non-functional SERT expression in
the baculovirus expression system and stable
mammalian cell lines
1. Baculovirus (Hi5 cells) 2. pCytTS-SERT 3.
T-Rex-SERT 4. 293-EBNA-Imi270
The same amount of functional SERT was loaded per
lane
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Stability of mammalian cell lines on continual
passaging
Day 3
293-EBNA-Imi270 Cell growth in the absence of
inhibitor
Day 12
Day 21
T-Rex-SERT
Day 90
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Conclusion
SERT is a complex membrane protein (1)
N-glycosylation essential for efficient
folding (2) Cholesterol is required for function
Because of this complexity, SERT expression is
best in mammalian cells using an inducible system
The T-Rex-SERT cell line was the best expression
system (1) Highest expression levels (2) No
unglycosylated, inactive SERT (3) Good cell
surface expression (4) Cell line is robust (5)
Suitable for large-scale fermentation
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References
Grisshammer Tate (1995) Q. Rev. Biophys. 28,
315-422 Tate Grisshammer (1996) Trends
Biotechnol. 14, 426-430 Tate (2001) FEBS Letters
504, 94-98 Tate et al., Biochim. Biophys. Acta
1610, 141-153