Expression vectors Useful for large-scale production of desired protein The basic requirements of an expression vector:

An origin of replication
A drug resistance marker (gene for ampicillin resistance, -lactamase).

An inducible promoter.
A multicloning site

The pUC Expression vector

Has following features The lac promoter (Plac) The associated operator (Olac) The lacI gene for the lac repressor protein The lacZgene produces an aminoterminal fragment of the -galactosidase protein. A polylinker (multiple cloning site). stretch of DNA within the lacZ' gene. contains restriction endonuclease sites.

The pUC Expression vector: -complementation

The lac promoter of the pUC plasmid can be induced using the lactose analogue isopropylthiol--D-galactoside. ("IPTG"). This will result in the expression of the lacZ' gene. This gene codes for an amino-terminal fragment of the -galactosidase protein The fragment is non-functional (it will not hydrolyze lactose or other -galactosides)

-complementation
The carboxy-terminal fragment of the protein if expressed separately is also non-functional But if peptide fragments from separate sources combine, they yield a functional galactosidase protein This is termed -complementation

Host bacteria enables -complementation

The lacZDM15 mutant bacteria have a deletion of the amino-terminal region of the lacZ gene. Have a non-functional -galactosidase protein Presence of extra-chromosomal element expressing the lacZ' peptide enables complementation The now complete -galactosidase hydrolyzes the galactoside, 5-bromo-4-chloro-3-indoyl-b-Dgalactoside ("X-gal"), to produce a dark blue color.

Host bacteria enables -complementation

lacZDM15 bacteria in the presence of lactose (or IPTG) and X-gal will appear a normal white-yellow color. The same bacteria which harbor the above pUC plasmid will be dark blue when IPTG and X-gal are present in the media (due to -complementation and a functional -galactosidase protein)

The pUC polylinker region A short stretch of DNA inserted within the lacZ' gene, just downstream from the start codon:

The different plink regions are all a multiple of 3 nucleotides in length, Therefore the lacZ' gene remains in-frame. The resulting lacZ'/plink peptide can still function in -complementation.

DNA fragment inserted into the plink region Three possible different reading frames after the insert (depending on the number of nucleotides in the inserted fragment) There is a 33% possibility that the downstream lacZ' gene will be translated in the correct reading frame Even then, the insertion at the start of the lacZ' protein will prevent complementation.

DNA fragment inserted into the plink region Therefore, most of the time, a DNA fragment in the plink region will abolish galactosidase function DNA inserts in the plink region can therefore be identified by growing the host bacteria on media containing IPTG and Xgal and looking for white-yellow colonies.

DNA fragment inserted into the plink region DNA coding for a gene of interest is inserted in the plink region in-frame with the lacZ' reading frame. The protein coded for by this gene can be expressed by induction with IPTG (i.e. using the Plac) The expressed protein will have as its amino-terminal sequence the first few amino acids of the -galactosidase gene

DNA fragment inserted into the plink region The expressed protein will have as its amino-terminal sequence the first few amino acids of the -galactosidase gene A fusion protein. The lac promoter is considered to be relatively weak, But the high copy number plasmid may result in a useful level of expression of the protein of interest

The pET vector system (Novagen)

It has the following important elements: Ampicillin resistance marker ColE1 origin of replication f1 origin of replication (allows single stranded vector to be produced when co-infected with M13 helper phage) lacI gene (lac repressor protein) T7 transcription promoter (specific for phage T7 RNA polymerase) lac operator region 3' to the T7 promoter multiple cloning site (polylinker region) downstream of the T7 promoter

The pET vector is a little different from the pUC vector: pUC uses the lac promoter and pET uses a promoter from phage T7 The phage T7 promoter is stronger than the lac promoter Phage T7 RNA polymerase specifically recognizes the T7 promoter region will not efficiently transcribe from other promoters The T7 promoter will not be efficiently transcribed by E. coli RNA polymerase

Where will the phage T7 RNA polymerase come from?

The pET system also involves a genetically engineered host bacteria. The host bacteria for the pET vector is typically E. coli strain BL(DE3) This strain has integrated into its chromosome the gene for T7 RNA polymerase

Where will the phage T7 RNA polymerase come from?

The T7 RNA polymerase in the host genome is constructed such that it is under the control of a lac promoter and operator Thus, induction by the lactose analogue, IPTG, causes the host to produce T7 RNA polymerase The E. coli host genome also carries the lacI (repressor) gene

Induction by IPTG results in:

De-repression of T7 RNA Pol gene on host chromosome

with subsequent production of this polymerase

De-repression of target gene under lac O regulation Transcription of target gene by T7 RNA Pol Thus, the system couples a strong promoter with tight

regulation (i.e. extremely low level of expression in the

repressed state)

 The pET vector is available with several polylinker sequences. They contain the same restriction sites, but differ in the reading frame leading into the pLink region:

The ggatcc site (BamH I) is the first restriction site in the polylinker. A gene of interest can be cloned in-frame with the transcribed region downstream from the T7 promoter. The expressed protein will be a fusion protein

The pTrcHis vector system (Invitrogen, Inc.) The important features are: Ampicillin resistance marker ColE1 origin of replication lacIq gene: is an up-regulated lac repressor mutation Trc promoter: a hybrid of the lac and trp promoters a stronger promoter than the lac promoter

 The pTrcHis vector system (Invitrogen, Inc.)

The lac operator region allows regulation by IPTG. 6 histidine residues in the amino terminal region of the translated protein, the "His tag" region. An enterokinase (EK) cleavage recognition sequence (asp asp asp asp lys , with cleavage after the lys residue) A polylinker region downstream from the EK site

Purpose of the His tag Histidine residues can coordinate to form a metal (Ni2+) binding site in a protein. Proteins with a His tag have affinity for metal chelating resins, and this characteristic can be used to selectively purify such proteins. Purpose of the EK cleavage site Presence of his tag may prevent normal function of the protein The His tag should be cleaved away from the protein The sequence "asp asp asp asp lys" is recognized and cleaved by enterokinase. This sequence is rare it is doubtful that the protein of interest will contain another such sequence

His tags/EK sites are typically introduced at the amino terminus of proteins, but can be introduced at the carboxyl terminus as well

The pL Expression system Another system making use of a phage promoter Gene is cloned downstream of Lambda PL promoter Regulated by the cI repressor. Using the temperature sensitive cI857 repressor allows control of gene expression by changing the growth temperature at 30 C the cI857 repressor is functional and it turns off expression of the gene, but at 42 C the repressor is inactivated so expression of the gene ensues. Alternatively, the wild-type cI gene can be placed under the control of another regulated promoter such as the PLAC promoter, Allows regulation by the addition of IPTG.

pL Expression System Offers the tightest transcriptional control of any bacterial expression system. Uses the strong pL promoter to drive expression The pL promoter is controlled by the lambda cI repressor protein,expressed in the E. coli host. The cI repressor gene is in the bacterial chromosome under the control of the tightly-regulated trp promoter. Expression is induced by the addition of tryptophan.

The pL Expression System is so tightly controlled Can be used to express even highly toxic proteins

General strategies for gene expression

Can express a protein in BL21 (DE3) After cloning in modified pET-vectors with the following tags and/or fusion partners: Tag N-terminal Histag Green Fluorescent protein glutathione-Stransferase (GST) C-terminal Histag Fusion Partner Purpose Affinity onto Ni2+ column Localise protein within cell Affinity chromatography with glutathione Affinity onto Ni2+ column

The choice of the host strains depends more on the nature of the heterologous protein. If the protein contains a high number of rare E. coli codons, express it in a strain that co-expresses the tRNAs for these rare codons. Strain
BL21 (DE3) CodonPlus-RIL BL21 (DE3) CodonPlus-RP Rosetta or Rosetta (DE3)

Rare codons
AGG/AGA (arginine), AUA (isoleucine) and CUA (leucine) AGG/AGA (arginine) and CCC (proline)

Supplier
Stratagene

Stratagene

AGG/AGA (arginine), CGG Novagen (arginine), AUA (isoleucine) CUA (leucine)CCC (proline), and GGA (glycine)

 If the protein contains one or more disulfide bonds, proper folding is stimulated in host strain with a more oxidizing cytoplasmic environment. Two strains are commercially available from Novagen:
Strain AD494 Properties Mutation in thioredoxin reductase (trxB) double mutant in thioredoxin reductase (trxB) and glutathione reductase (gor)

Origami

If the protein is toxic to the cell,

Express in a strain containing the pLysS or pLysE vector tightens regulation of expression systems using the T7 promoter. These vectors express lysozyme, which binds to and inactivates T7 RNA polymerase.

If the protein is membrane-bound,

Express in mutant strains C41 (DE3) and C43 (DE3) could improve expression levels. Miroux, B. & Walker, J.E. (1996) J. Mol. Biol. 260, 289298.

Careful when using ampicillin!. -lactamase, is secreted into the medium where it hydrolyses all of the ampicillin. This point is reached when the culture is barely turbid. Thereafter, cells that lack the plasmid will not be killed and could overgrow the culture
Some possible solution are: Grow overnight cultures at 30C or lower. Spin overnight cultures and resuspend the pellet in fresh medium to remove -lactamase. Use the more stable carbenicillin instead of ampicillin.

After Inoculation of the main culture

Incubate until OD600 reaches 0.4-1. (0.6 is recommended LB-medium). To increase the growth rate, cultures at 37C until the OD for induction is

reached.
Then cool to the induction temperature in ice-water. For good aeration, don't use more medium than 20% of the total flask volume.

Induction of protein expression. Protein expression is induced by Addition of the proper inducer Changing the growth conditions.
From this point on, the cells will use most of their resources for the production of the target protein.

Promoter Trc (hybrid)

araBAD PL T7-lac

Induction IPTG
Temp. shift IPTG

Typical 0.2mM
From 37 to 42 0.2mM

Range 0.05-2.0mM
0.002-0.4%

1-Arabinose 0.2%

0.05-2.0mM

After induction
The cultures are incubated from 3 hours to overnight depending on the induction temperature.

Incubation temperature 15C 20C 25C 30C 37C

incubation time
overnight overnight overnight 5-6 h 3-4 h

Harvesting of the cell pellet centrifugation (20 min at 6000 g). Cell pellets are stored at -20C.

Expression hosts Allow the expressed protein to accumulate. Some expressed proteins may not be folded correctly. misfolded proteins may be rapidly proteolyzed in the host bacterium Host proteases may selectively cleave precursor forms of expressed proteins to produce mature active forms, which may not be desirable. OmpT and lon genes code for proteases which can degrade expressed proteins. Two common mutations in E. coli to eliminate the action of host proteases includes the OmpT- and lon- strains. E. coli strain BL(DE3) is both OmpT- and lon-