Expression systems Lecture 7 Designing vectors and making protein Few slides by David Tscharke @ RSB

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 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

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

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

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)

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 Showing the rRNA of the 30S subunit after stripping most proteins Shine-Dalgarno helix involving 16S RNA

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, 11989 (2000)

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 way Can 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 1973 No Nobel prize Herby got filthy rich…

Protein overexpression Induction with IPTG Autoinduction Cell-free

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.

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

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

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)

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!

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!

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

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.”

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