1. Expression systems Lecture 7 Designing vectors and making protein
Few slides by David RSB

2. Lecture overview Ingredients of an overexpression system (cloning)
Growing E. coli
Induction of protein expression

3. The ultimate overexpression system
High copy-number plasmid
Controlled by the origin of replication
Efficient mRNA production
E. coli strain with T7 RNA polymerase (faster than E. coli RNAP)
Limited protease activity
E. coli strain deficient in proteases
Efficient for heterologous genes
Additional plasmid with tRNA genes for codons rare in E. coli
Synthetic gene (50 cents per bp)

4. Most popular expression system
Enhanced mRNA production
E. coli strain BL21(DE3)
Contains the T7 RNA polymerase in genome under control
of the lacUV5 promoter
Induction with IPTG starts expression of T7 RNAP
T7 RNAP transcribes genes under control of the T7 promoter
Strain is deficient in lon and ompT proteases
Immune to bacteriophage 21
“T7 vector”
Plasmid with gene of interest preceded by the T7 promoter
and followed by the T7 terminator
High copy-number plasmid
Helpful but less effective than expression of T7 RNAP

5. T7 RNAP versus E. coli RNAP
459 kDa
6 subunits (a2bb’ws)
s factor dissociates after initiation
Transcribes from many different promoters
T7 RNAP
99 kDa
Single polypeptide chain
Extremely promoter specific
EM of E. coli RNAP on DNA
T7 RNAP
with DNA

6. E. coli promoters E. coli RNAP binds to the -35 and -10 regions
Sense sequences of selected E. coli promoters
E. coli RNAP binds to the -35 and -10 regions
Transcription starts at the initiation site

7. T7 promoter Small binding site T7 RNAP binds with very high affinity
Not recognized by E. coli RNAP
Pattern required by T7 RNAP to function
Schneider & Stephens, Nucl. Acids Res. 18, 6097 (1990)

8. RNAP transcribes until it meets a terminator

9. E. coli terminator 1) GC-rich hairpin in mRNA, followed by 7-10 U’s
“transcription bubble”
site for incoming NTP
2) In half the cases, by Rho factor
Helicase that binds to mRNA
nucleotide recognition sequence

10. T7 terminator
GC-rich hairpin in mRNA, followed by 7-10 U’s
e.g. in gene 10 (coat protein) of the T7 bacteriophage:
AACCCCTTGG GGCCTCTAAA CGGGTCTTGA GGGGTTTTTT G
<<<<<<< < << >> > > >>>>>>
Non-perfect base pairing in the hairpin is OK
E. coli terminators work fine
Actually, no terminator often works too…

11. Ribosome binding site (Shine-Dalgarno)
Ribosome binding site (RBS)
About 10 nucleotides prior to AUG start codon
Complementary to 16S rRNA of the ribosome
Promotes binding of the ribosome to the mRNA

12. Ribosome with mRNA Showing the rRNA of the 30S subunit
after stripping most proteins
Shine-Dalgarno helix
involving 16S RNA

13. A real-life vector Perfect insert has NdeI site at 5’ end
T7 terminator
Resistance gene
bla: “b-lactamase ampicillin”
Perfect insert has
NdeI site at 5’ end
EcoRI or HindIII
site at 3’ end
T7 promoter
RBS
Start codon
Neylon et al. Biochemistry 39, (2000)

14. Controls for cloning A typical cloning experiment has: Several steps
Takes several days
You can’t easily check each step
When things go right, it’s quick
Tempting to forget about controls
What you can test:
The quality of enzyme function (only sometimes)
Can always test enzymes
Can’t always test them with specific DNA
The competency of your cells for transformation
Re-ligation of vector

15. The most important control!
Vector only control
Even if you do nothing else…
Full protocol without insert +
transform
Tells you how many colonies can come from the vector alone = ‘background’
May be re-ligated vector
May be some vector that didn’t get cut (need only pg)
May be DNA contamination

16. What is ‘cloning strategy’?
There are often several ways to make a clone
These are the different ‘strategies’
Each strategy has strengths and weaknesses
A strategy must go all the way
Can be considered to have five main steps:
Prepare insert (may include adding RE site using PCR)
Prepare vector
Ligate
Transform
Screen

17. Summary I Overexpression vector needs
promoter and terminator for transcription
ribosome binding site (Shine-Dalgarno) for translation
Using PCR to generate an insert with easy-to-clone ends (e.g. suitable restriction enzyme sites) is a very versatile method
But beware of the error rate of PCR
Never forget the ‘vector alone’ control in cloning

18. A bit of history… Stanley Cohen Herbert Boyer
These men made the first molecular clone
In which decade did they do it?
Did they get a Nobel prize?

19. A bit of history… Stanley Cohen Herbert Boyer 1973 No Nobel prize
Herby got filthy rich…

20. Protein overexpression
Induction with IPTG
Autoinduction
Cell-free

21. E. coli – rich medium Luria-Bertani broth (“LB”) Industry standard
Made from
Tryptone (peptides)
Yeast extract (water soluble
fraction of self-digested cells
- vegemite)
NaCl
Usually also includes
Vitamins
Trace elements (metals)
Autoclave at 121 oC
to sterilize
Tryptone is made from
casein (milk protein) by
digestion with trypsin.

22. E. coli – minimal medium M9 minimal medium
For labelling with expensive isotopes (15N, 13C, 2H)
Defined carbon source
e.g. glucose, glycerol, acetate, etc.
Defined nitrogen source
NH4Cl
Defined salts
NaCl, Na2HPO4, KH2PO4, MgSO4, CaCl2
Autoclave at 121 oC to sterilize

23. Growth curve
The cell density is measured by absorption of light at 600 nm
wavelength (optical density, “OD600”)
OD600 is directly proportional to the cell density
Growth curves of different E. coli strains
Maximal ribosome
concentration
Induce at this point
plateau {
exponential
growth

24. Induction Start of protein overexpression
lac operon controls expression of T7 RNAP in E. coli BL21(DE3)
lac operon is a specific sequence of DNA
BL21(DE3) expresses a low level of lac repressor
lac repressor is a tetrameric DNA-binding protein
lac repressor binds to lac operon
Silencing the following gene (competition with RNAP)
IPTG binds to lac repressor
Binding causes a change in relative orientation of lac repressor
molecules, abolishing cooperative binding to the DNA
RNAP gains access to promoter when lac repressor leaves

25. Lac repressor, lac operon, IPTG
Isopropyl β-D-1-thio-galactopyranoside
lac operon
IPTG binding site
Part of the other dimer in
the lac repressor tetramer
lac repressor (dimer)

26. Autoinduction
Method by F.W. Studier (2005) Protein Expr. Purif. 41, 207.
Based on ability of certain media to induce protein expression in E. coli when cells reach saturation
Once glucose has been used up, lactose in the medium is converted to allo-lactose that releases lac repressor
Auto-induction can be regulated by adjusting glucose/lactose levels in media
No need to monitor OD600
2-3 times higher OD600 can be reached
Protein expressed while you sleep!

27. Cell-free protein synthesis
Using the E. coli cytosol to make proteins
strip E. coli of its cell wall by shearing (pushing cells through
a small pore turns them inside-out)
Spin down genomic DNA and cell debris
Place cytosol in a dialysis bag
~ 1 ml, contains all soluble E. coli enzymes
~ 10 ml, contains ATP, nucleotides, amino acids
Add DNA, T7 RNAP, tRNA to reaction volume – get protein!

28. Cell-free protein synthesis
Typically 1 mg protein/ml reaction mixture
Good for
Proteins toxic to E. coli
Membrane proteins (detergents can be added to solubilize
the proteins as they are made)
Proteins from expensive labelled amino acids (because the
proteins are produced in a small volume and the natural
metabolism is defunct)
Fast
Proteins can be made from linear PCR-amplified DNA in
a few hours

29. Summary II T7 overexpression systems are the gold standard
Induction with IPTG
Classical
Autoinduction
Lazy
Cell-free
Speedy

30. The last word… Murphy’s law “If anything can go wrong, it will”
Stapp’s paradox
“The universal aptitude for ineptitude makes any human accomplishment an incredible miracle.”

31. Prac this afternoon in T4: 2 pm sharp
Lab coat and safety glasses
Risk assessment
An exercise book that will be your Log book
Read the first day of the prac and prepare a one page flow chart of the day’s experiments
Team up in pairs