www.t-cellbiology.org/teaching.htm

Simon Davis, Ed Evans: 21336

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

Expression cloning: lambda libraries

plaque cDNA library plaque screen

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

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

Expression cloning: 2-hybrid systems

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

Bacterial expression

Recommended: pET vectors- T7 promoter-based systems

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

Recommended:- EF1-based expression vectors

Transient expression

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

Is my protein any good?

Good signs, cont.- the protein is non-aggregated according to

gel filtration - the absolute key for doing BIAcore experiments and structural studies properly