Expression systems

Lecture 7

Few slides by David Tscharke @ RSB

Designing vectors and making protein

Lecture overview

Ingredients of an overexpression system (cloning)

Growing E. coli

Induction of protein expression

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)

Most popular expression systemEnhanced mRNA productionE. 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

T7 RNAP versus E. coli RNAPE. coli RNAP-459 kDa -6 subunits (2’) 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

E. coli promotersSense sequences of selected E. coli promoters

E. coli RNAP binds to the -35 and -10 regionsTranscription starts at the initiation site

T7 promoter

Pattern required by T7 RNAP to function

Schneider & Stephens, Nucl. Acids Res. 18, 6097 (1990)

- Small binding site- T7 RNAP binds with very high affinity- Not recognized by E. coli RNAP

RNAP transcribes until it meets a terminator

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-80-100 nucleotide recognition sequence

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…

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

Ribosome with mRNA

Shine-Dalgarno helixinvolving 16S RNA

Showing the rRNA of the 30S subunitafter stripping most proteins

A real-life vector

Neylon et al. Biochemistry 39, 11989 (2000)

T7 promoter

T7 terminator

RBS

Start codon

Resistance genebla“-lactamase ampicillin”

Perfect insert has-NdeI site at 5’ end-EcoRI or HindIII site at 3’ end

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

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

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 wayCan be considered to have five main steps:-Prepare insert (may include adding RE site using PCR)-Prepare vector -Ligate-Transform-Screen

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

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?

A bit of history…

Stanley Cohen Herbert Boyer

1973No Nobel prize

Herby got filthy rich…

Protein overexpression

Induction with IPTG

Autoinduction

Cell-free

E. coli – rich mediumLuria-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 oCto sterilize

Tryptone is made fromcasein (milk protein) bydigestion with trypsin.

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

Growth curveThe cell density is measured by absorption of light at 600 nmwavelength (optical density, “OD600”)-OD600 is directly proportional to the cell density

Growth curves of different E. coli strains

http://www.vli-research.com/silantes_labeled.htm

{exponentialgrowth

plateau

Maximal ribosomeconcentrationInduce at this point

Induction

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

Start of protein overexpression

Lac repressor, lac operon, IPTGlac operon

lac

repr

esso

r (di

mer

)

Part of the other dimer in

the lac repressor tetramer

IPTG binding site

Isopropyl β-D-1-thio-galactopyranoside

AutoinductionMethod 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!

Cell-free protein synthesisUsing 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 acidsAdd DNA, T7 RNAP, tRNA to reaction volume – get protein!

Cell-free protein synthesisTypically 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

Summary II

T7 overexpression systems are the gold standard

Induction with IPTG-Classical

Autoinduction-Lazy

Cell-free-Speedy

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.”

•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

Prac this afternoon in T4: 2 pm sharp