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Methods Course, October 25th
Expression cloning
1. In bacteria
2. In mammalian cells
3. Yeast 2-hybrid screens
Protein expression
1. Why express proteins at all?
2. How to decide on an expression strategy
3. The methods
- bacterial expression: the pET system
- mammalian expression (transient, stable)
4. Judging protein quality
The BIAcore (Ed Evans)
Methods Course, October 25th
Expression cloning: lambda libraries
Principle
1. Construct cDNA library in
bacteriophage expression vector, e.g.
“ zap” or gt11
2. Transduce (infect) bacterial host
3. Plate out, allow plaques to form
4. Transfer expressed protein to
membrane
5. Screen proteins with antibody
(Western)
6. Isolate clone from library
Advantage1. Not restricted to monomers or
homodimers Disadvantages1. Most proteins do not fold spontaneously
in bacteria, so need non-conformation sensitive antibodies (e.g. polyclonal antisera)
2. So generally can’t use ligands to screen3. Will not automatically select full-length
clones
Expression cloning: lambda libraries
Principle1. Transfect COS-1 cells with cDNA
library in CDM82. Pan on antibody coated dish3. Isolate plasmid from bound cells,
re-transfect4. Repeat cycle 3-4 times5. Transfect individual mini-preps to
identify gene, then sequence
Expression cloning with CDM8
Expression cloning with CDM8
Y Y Y Y Y Y YCos-1 cell
antibody
transiently expressed proteins
Y Y Y Y Y Y Y
sequence clone
Expression cloning with CDM8
Advantages1. Can use monoclonal antibodies2. Isolates full length cDNAs3. Can potentially use ligands
Disadvantages1. Only useful for monomers,
homodimers etc
Advantage1. Designed for identifying ligand-
receptor pairs
Disadvantages1. Only useful for protein pairs that fold
well in the cytoplasm2. Likely to require a high affinity
interaction of proteins X and Y3. Said to be difficult
Expression cloning: 2-hybrid systems
Why express and study proteins?
1. Proteins are of fundamental interest: biological systems are all about protein recognition
2. An understanding of immunological phenomena increasingly depends on understanding how proteins behave
3. Can expect hard answers to scientific questions: is this how my protein looks?
4. Modern immunology is reagent-driven so the choice of protein can set the research agenda
5. This can provide many opportunities for collaboration (i.e. lots of papers)
Why express and study proteins?
The expression strategy
Bacterial expression (e.g. pET vectors)- fast - often very large amounts
Your protein Cytosolic?
The expression strategy
1. Bacterial re-folds- yields can be low (~1%)- refold conditions generally differ for each protein- sparse-matrix screens are available to help
Your proteinSecreted or
membrane bound?
The expression strategy
2. Bacterial secretion
systems- e.g. pET-12a,b,c vectors- yields often very low
Your proteinSecreted or
membrane bound?
The expression strategy
1.Yeast (e.g. Pichia)- fast- very high yields- metabolic labelling
(NMR)- deglycosylation
possible- poor folding of e.g. IgSF proteins
Your proteinSecreted or
membrane bound?
needs to be
glycosylated or don’t want to refold?
The expression strategy
2. Baculovirus- can be very slow- modest yields: 1-5
mg/l- very good for some
proteins e.g. MHC II
Your proteinSecreted or
membrane bound?
needs to be
glycosylated or don’t want to refold?
The expression strategy
3. Mammalian cells(e.g. CHO K1 cells)
- moderately fast- very high yields
potentially (<400 mg/l)- sugars can be
removed- (Lec3.2.8.1 cells)- transient or stable
Your proteinSecreted or
membrane bound?
needs to be
glycosylated or don’t want to refold?
Bacterial expression
Basic features
- proteins are expressed in the
cytoplasm or secreted into the
periplasmic space
- periplasmic expression levels are very
low
- very few cell surface or secreted
proteins fold in the cytosol
- so most proteins of interest form
inclusion bodies and have to be
refolded
Mammalian expression
Basic features- expressed proteins are generally secreted,
but can be put on the cell surface or made intracellularly
- soluble expression is achieved by inserting stop codon immediately before the TM domain
- proteins are glycosylated; refolding unnecessary
- the more “intact” the protein, the better- fusion proteins, his-tagged proteins can be
made
Transient expression
Advantages- transient expression takes 3-5 days- excellent for testing constructs- various fusion partners- transfection with CaPO4 or lipids (fast)Disadvantage- beware of Fc fusion proteins - Fc folds very
efficiently, possibly taking mis-folded protein with it
Stable expression
The GS system- CHO cells transfected with CaPO4 or lipids- selection is via the glutamine synthetase
(GS) gene- CHO cells have their own GS gene and can
be killed with GS inhibitor, methionine sulphoximine
- cells with extra GS from the plasmid survive higher levels of MSX than the mocks
- expression is driven by strong hCMV promoter
Stable expression
The GS system, cont.- selection takes 2 weeks- potentially prodigious expression levels - Can make enough protein to thoroughly
confirm that it’s OK- mutant CHO cells can be used to alter
glycosylation, e.g. Lec3.2.8.1 cells
- NO DISADVANTAGES
AmpR
hCMV promoter
glutamine synthetasegene
SV40 promoter
poly A
expressed protein
5 lclone4A tcs
5 l control
tcs2 g CD4
400 mgs/litre =10 x “Harry Ward” units
The glutamine synthetase-based gene expression system
pEE14.hcmv-GS10.4 kb
Lec3
.2.8
.1
+ e
ndo
H
CHO-K
1Le
c3.2
.8.1
Expression of rat sCD2 for structural studies in CHO mutant Lec3.2.8.1 cells
Deglycosylated sCD2 crystals
Is my protein any good?
Good signs- it’s expressed at high levels- if cys-containing, it runs at the right size on
non-reducing SDS-PAGE- the protein is stable/active for days/weeks at
4ºC- the protein binds mAbs stoichiometrically
(Westerns and ELISAs are not suitable for this)
- the protein is soluble at high concentrations