Vector Promoter Selection; Tags and Fusion partners Protease cleavage site Origin Supplier
pALTER-Ex1 T7 Tet none none
Promega
pALTER-Ex2 T7 Tet none none
Promega
pBAD/His araBAD Amp N-His EK pUC Invitrogen
pBAD/Myc-His araBAD Amp C-His none pUC Invitrogen
pBAD/gIII araBAD Amp leader sequence
C-His
none ColE1 Invitrogen
pCal-n T7-lac Amp N-CBP Thr ColE1 Stratagene
pCal-n-EK T7-lac Amp N-CBP Thr
EK  
ColE1 Stratagene
pCal-c T7-lac Amp C-CBP Thr ColE1 Stratagene
pCal-Kc T7-lac Amp C-CBP Thr ColE1 Stratagene
pcDNA 2.1 T7 Amp none none pUC Invitrogen
pDUAL T7-lac Kan C-CBP Thr ColE1 Stratagene
pET-3a-c T7 Amp none none pBR322 Novagen
pET-9a-d T7 Kan none none pBR322 Novagen
pET-11a-d T7-lac Amp none none pBR322 Novagen
pET-12a-c T7 Amp none none pBR322 Novagen
pET-14b T7 Amp N-His Thr pBR322 Novagen
pET-15b T7-lac Amp N-His Thr pBR322 Novagen
pET-16b T7-lac Amp N-His Xa pBR322 Novagen
pET-17b T7 Amp none none pBR322 Novagen
pET-19b T7-lac Amp N-His EK pBR322 Novagen
pET-20b(+) T7 Amp signal sequence
C-His
none pBR322 Novagen
pET-21a-d(+) T7-lac Amp C-His none pBR322 Novagen
pET-22b(+) T7-lac Amp signal sequence
C-His
none pBR322 Novagen
pET-23a-d(+) T7 Amp C-His none pBR322 Novagen
pET-24a-d(+) T7-lac Kan C-His none pBR322 Novagen
pET-25b(+) T7-lac Amp signal sequence
C-His
none pBR322 Novagen
pET-26b(+) T7-lac Kan Signal sequence
C-His
none pBR322 Novagen
pET-27b(+) T7-lac Kan signal sequence
C-His
none pBR322 Novagen
pET-28a-c(+) T7-lac Kan N-His
C-His
Thr pBR322 Novagen
pET-29a-c(+) T7-lac Kan C-His Thr pBR322 Novagen
pET-30a-c(+) T7-lac Kan N-His
C-His
Thr
EK  
pBR322 Novagen
pET-31b(+) T7-lac Amp C-His none pBR322 Novagen
pET-32a-c(+) T7-lac Amp internal His
C-His
Thr
EK  
pBR322 Novagen
pET-33b(+) T7-lac Kan internal His
C-His
Thr
EK  
pBR322 Novagen
pET-34b(+) T7-lac Kan N-CBDclos
C-His
Thr
EK  
pBR322 Novagen
pET-35b(+) T7-lac Kan N-CBDclos
C-His
Thr
Xa  
pBR322 Novagen
pET-36b(+) T7-lac Kan signal sequence
N-CBDcenA
C-His
Thr
EK  
pBR322 Novagen
pET-37b(+) T7-lac Kan signal sequence
N-CBDcenA
C-His
Thr
Xa  
pBR322 Novagen
pET-38b(+) T7-lac Kan signal sequence
C-CBDcex
C-His
Thr pBR322 Novagen
pET-39b(+) T7-lac Kan (signal sequence)
DsbA
N-His
C-His
Thr
EK  
pBR322 Novagen
pET-40b(+) T7-lac Kan (signal sequence)
DsbC
N-His
C-His
Thr
EK  
pBR322 Novagen
pET-41a-c(+) T7-lac Kan GST
N-His
C-His
Thr
EK  
pBR322 Novagen
pET-42a-c(+) T7-lac Kan GST
N-His
C-His
Thr
Xa  
pBR322 Novagen
pET-43a-c(+) T7-lac Kan NusA
N-His
C-His
Thr
EK  
pBR322 Novagen
pETBlue-1 T7-lac Amp C-His none pUC Novagen
pETBlue-2 T7-lac Amp C-His none pUC Novagen
pETBlue-3 T7-lac Amp N-His
C-His
Thr
EK  
pUC Novagen
pGEMEX-1 T7 Amp T7 gene 10 none
Promega
pGEMEX-2 T7 Amp T7 gene 10 none
Promega
pGEX-1lT tac Amp GST Thr pBR322 Pharmacia
pGEX-2T tac Amp GST Thr pBR322 Pharmacia
pGEX-2TK tac Amp GST Thr
EK  
pBR322 Pharmacia
pGEX-3X tac Amp GST Xa pBR322 Pharmacia
pGEX-4T tac Amp GST Thr pBR322 Pharmacia
pGEX-5X tac Amp GST Xa pBR322 Pharmacia
pGEX-6P tac Amp GST Pre pBR322 Pharmacia
pHAT10/11/12 lac Amp N-HAT EK pUC Clontech
pHAT20 lac Amp N-HAT EK pUC Clontech
pHAT-GFPuv lac Amp N-HAT
N-GFPuv
EK pUC Clontech
pKK223-3 tac Amp none none pBR322 Pharmacia
pLEX PL  Amp none none pUC Invitrogen
pMAL-c2X tac Amp N-MBP Xa ColE1 NEB
pMAL-c2E tac Amp N-MBP EK ColE1 NEB
pMAL-c2G tac Amp N-MBP Gen ColE1 NEB
pMAL-p2X tac Amp N-MBP Xa ColE1 NEB
pMAL-p2E tac Amp N-MBP EK ColE1 NEB
pMAL-p2G tac Amp N-MBP Gen ColE1 NEB
pProEX HT trc Amp N-His TEV
Life Technologies
pPROLar.A lac-ara1 Kan N-c-Myc EK p15A Clontech
pPROTet.E LtetO-1 Cam N-c-Myc EK ColE1 Clontech
pQE-9 T5-lac Amp N-His none ColE1 Qiagen
pQE-16 T5-lac Amp C-His none ColE1 Qiagen
pQE-30/31/32 T5-lac Amp N-His none ColE1 Qiagen
pQE-40 T5-lac Amp N-His
N-DHFR
none ColE1 Qiagen
pQE-60 T5-lac Amp C-His none ColE1 Qiagen
pQE-70 T5-lac Amp C-His none ColE1 Qiagen
pQE-80/81/82L T5-lac Amp N-His none ColE1 Qiagen
pQE-100 T5-lac Amp N-His none ColE1 Qiagen
pRSET T7 Amp N-His EK ColE1 Invitrogen
pSE280 trc Amp none none pUC Invitrogen
pSE380 trc Amp none none pUC Invitrogen
pSE420 trc Amp none none pUC Invitrogen
pThioHis trc Amp His-Patch Trx EK ColE1 Invitrogen
pTrc99A trc Amp none none pBR322 Pharmacia
pTrcHis trc Amp N-His EK pUC Invitrogen
pTrcHis2 trc Amp C-His none pUC Invitrogen
pTriEx-1 T7 Amp C-His none pUC Novagen
pTriEx-2 T7-lac Amp N-His
C-His
Thr
EK  
pUC Novagen
pTrxFus PL  Amp Trx EK ColE1 Invitrogen

The basic architecture of an E. coli expression vector is shown in the figure below and contains the following features: 

Selectable marker. In the absence of selective pressure plasmids are lost from the host. Especially in the case of very high copy number plasmids and when plasmid-borne genes are toxic to the host or otherwise significantly reduce its growth rate. The simplest way to address this problem is to express from the same plasmid an antibiotic-resistance marker and supplement the medium with the appropriate antibiotic to kill plasmid-free cells. The most used antibiotics and their effective concentrations are listed in table 1.

The use of ampicillin requires special care. The selectable marker, b-lactamase, is secreted into the medium where it hydrolysis all of the ampicillin. This point is already reached when the culture is barely turbid. From here on, cells that lack the plasmid will not be killed and could overgrow the culture. Some possible solution are:

  • grow overnight cultures at 30°C or less.
  • spin overnight cultures and resuspend the pellet in fresh medium to remove the produced b-lactamase.
  • use the more stable carbenicillin instead of ampicillin.

Regulatory gene (repressor). Many promoters show leakiness in their expression i.e. gene products are expressed at low level before the addition of the inducer. This becomes a problem when the gene product is toxic for the host. This can be prevented by the constitutive expression of a repressor protein.

The lac-derived promoters are especially leaky. These promoters can be controlled by the insertion of a lac-operator sequence downstream the promoter and the expression of the lac-repressor by host strains carrying the lacIq allele (for medium copy number plasmids) or from the same or a helper plasmid (for higher copy number plasmids). Alternatively, repression can be achieved by the addition of 1% glucose to the culture medium.

Origin of replication. The origin of replication controls the plasmid copy number.

Promoter. The promotor initiates transcription and is positioned 10-100 nucleotides upstream of the ribosome binding site. The ideal promoter exhibits several desirable features:

  • It is strong enough to allow product accumulation up to 50% of the total cellular protein.
  • It has a low basal expression level (i.e. it is tightly regulated to prevent product toxicity).
  • It is easy to induce.

An extensive list of possible promoters is given in table 2. The most used promoters are indicated in red.

Transcription terminator. The transcription terminator reduces unwanted transcription and increases plasmid and mRNA stability.

Shine-Delgarno sequence. The Shine-Dalgarno (SD) sequence is required for translation initiation and is complementary to the 3'-end of the 16S ribosomal RNA. The efficiency of translation initiation at the start codon depends on the actual sequence. The concensus sequence is: 5'-TAAGGAGG-3'. It is positioned 4-14 nucleotides upstream the start codon with the optimal spacing being 8 nucleotides. To avoid formation of secondary structures (which reduces expression levels) this region should be rich in A residues.

Start codon. Initiation point of translation. In E. coli the most used start codon is ATG. GTG is used in 8% of the cases. TTG and TAA are hardly used.

Tags and fusion proteins. N- or C-terminal fusions of heterologous proteins to short peptides (tags) or to other proteins (fusion partners) offer several potential advantages:

  • Improved expression. Fusion of the N-terminus of a heterologous protein to the C-terminus of a highly-expressed fusion partner often results in high level expression of the fusion protein.
  • Improved solubility. Fusion of the N-terminus of a heterologous protein to the C-terminus of a soluble fusion partner often improves the solubility of the fusion protein.
  • Improved detection. Fusion of a protein to either terminus of a short peptide (epitope tag) or protein which is recognized by an antibody or a binding protein (Western blot analysis) or by biophysical methods (e.g. GFP by fluorescence) allows for detection of a protein during expression and purification.
  • Improved purification. Simple purification schemes have been developed for proteins fused at either end to tags or proteins which bind specifically to affinity resins (see table 3 and references therein).

Protease cleavage site. Protease cleavage sites are often added to be able to remove a tag or fusion partner from the fusion protein after expression. Most commonly used proteases are listed in table 4. However, cleavage is rarely complete and often additional purification steps are required.

Multiple cloning site. A series of unique restriction sites that enables you to clone your gene of interest into the vector.

Stop codon. Termination of translation. There are 3 possible stop codons but TAA is preferred because it is less prone to read-through than TAG and TGA. The efficiency of termination is increased by using 2 or 3 stop codons in series.