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 Tet expression system
Features and benefits
- High-level expression in E. coli with pASK-IBA vectors
- Tightly regulated expression due to the tet promoter
- Enhanced stability of cytotoxic genes
- Universal cloning strategy with one restriction enzyme
- N- or C-terminal Strep-tag II fusion
- N- or C-terminal 6xHistidine-tag fusion
- Strep-tag II/6xHistidine-tag - double tag vectors
- Cytosolic or periplasmic expression
- Inexpensive induction with anhydrotetracycline
- Ampicillin or chloramphenicol resistance
- TEV protease cleavage site
- all vectors at a glance
Principle and properties
pASK-IBA vectors work with the tightly regulated tet promoter. The tet repressor is encoded on the pASK-IBA plasmids and is constitutively expressed from the b-lactamase or the chloramphenicol acetyl transferase promoter, respectively. This special arrangement guarantees a balanced stochiometry between repressor molecules and plasmid copy number. Expression of the foreign gene is stringently repressed until efficient chemical induction with a low concentration of anhydrotetracycline. In contrast to the lac promoter - which is leaky, susceptible to catabolite repression (cAMP-level, metabolic state), and influenced by chromosomally encoded repressor molecules - the tetA promoter/operator is tightly controlled and not functionally coupled to any cellular regulation mechanisms or genetic background.
As a consequence, special E. coli strains or extra plasmids are not required and a broad range of culture media and conditions can be used. For example, glucose minimal media and even the XL1-Blue bacterial strain, which carries an episomal copy of the tetracycline resistance gene, can be used for expression. The pASK-IBA expression system is stable under many conditions, including fermentation, and is easy-to-handle.
Further elements of the vectors are a tandem ribosome binding site (RBS) which ensures efficient initiation of translation, the strong terminator of the lipoprotein gene in order to prevent read-through, the intergenic region of the bacteriophage f1 which provides a means for preparing ssDNA and a b-lactamase or chloramphenicol acetyl transferase gene*. The vectors do not mediate resistance against tetracycline.
*Using cloning vectors with b-lactamase resistance gene may be associated with some limitations since ampicillin is degraded quite fast in bacterial culture medium. Therefore, we are now offering our Strep-tag II vectors pASK-IBA2C to pASK-IBA7C with chloramphenicol resistance instead of ampicillin resistance.
Reference:
Skerra, A. (1994). Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli. Gene, 151, 131-135.
The following E. coli strains have already been used successfully for Tet expression with our pASK-IBA vectors: JM83, WK6, B, BL21, MG1655, W3110, BL21(DE3), BLR(DE3), XL1-Blue, BL21-CodonPlusTM-RIL
For secretion, we recommend JM83. For cytoplasmic expression E. coli B strains are recommended, since they lack the lon protease and the ompT outer membrane protease that can degrade proteins during purification (Grodberg and Dunn, 1988, J.Bacteriol. 170, 1245).
Please note that we are not aware of an E. coli strain that is incompatible with the Tet expression system.
Anhydrotetracycline: Inducer for tetA Promoter
The E. coli expression cassette of the Strep-tag/Strep-Tactin over-expression system is under transcriptional control of the tetA promoter/operator and repressor. The promoter is induced by a low concentration of anhydrotetracycline (AHT) saving costs and minimizing the antibacterial influence of AHT. Degenkolb et al. (1991) have shown, that AHT binds 35-times tighter than tetracycline to the tet repressor.
Literature:
Degenkolb J, Takahashi M, Ellestad GA, Hillen W, 1991:Antimicrob. Agents Chemother. 35, No 8, 1591-1595 Structural requirements of tetracycline-Tet repressor interaction: Determination of equilibrium binding constants for tetracycline analogues with the Tet repressor.
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Order information
see also "how to order"
| product |
amount |
cat. no. |
order & prices |
| Anhydrotetracycline |
50 mg (for 250 liter E.coli culture) |
2-0401-001 |
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| Anhydrotetracycline |
25 mg |
2-0401-002 |
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Literature for anhydrotetracycline:
Degenkolb J, Takahashi M, Ellestad GA, Hillen W, 1991:Antimicrob. Agents Chemother. 35, No 8, 1591-1595. Structural requirements of tetracycline-Tet repressor interaction: Determination of equilibrium binding constants for tetracycline analogues with the Tet repressor.
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T7 expression system
Features and benefits
- High-level expression by bacteriophage T7 promoter with pPR-IBA vectors
- High-level transcription by T7 RNA polymerase in BL21 strains
- High-level expression of non-toxic proteins
- Induction by IPTG
- N- or C-terminal Strep-tag II
or One-STrEP-tag
- Suitable for in vitro transcription/translation
Principle and properties
The Tet promoter is of medium strength which leads to high level expression of certain proteins depending e.g. on their folding rate and stability - characteristics which can hardly be predicted. Some proteins, however, can only be expressed at high level if transcribed by a stronger promoter. In such cases we recommend the use of the T7 promoter.
The T7 expression system is encoded on the pPR-IBA vectors. The system uses the T7 promoter and T7 RNA polymerase for high-level transcription of the gene of interest. Expression of the target genes is induced by providing a source of T7 RNA polymerase in the host cell. This is accomplished by using E. coli BL21 which contains a chromosomal copy of the T7 RNA polymerase gene. The latter is under control of the lacUV5 promoter which can be induced by IPTG.
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Remarks on protein expression in E. coli
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Formation of disulfide bonds
Some vectors provide a N-terminal fusion of the ompA signal peptide which mediates the secretion of the recombinant protein to the periplasmic space of E. coli. There, the signal peptide is selectively cleaved by the E. coli signal peptidase. The secretion strategy is essential for the functional production of proteins containing structural disulfide bonds that are often present in naturally secreted proteins. The reducing conditions in the cytoplasm of E. coli prevent disulfide bond formation which can lead to aggregation or degradation of unfolded polypeptides.
Periplasmic secretion as a first purification step
Furthermore, periplasmic secretion separates the recombinant protein from cytosolic proteases. Since the E. coli outer membrane can be selectively degraded by mild treatment (EDTA, lysozyme etc.) the spheroplasts containing the cytosolic components can be easily removed by centrifugation.
Addition of active substances
In addition, the periplasmic space is accessible to molecules < 600 Da allowing to influence folding or stability of the recombinant protein during expression by adding active substances to the culture media (e.g. redox components, non-metabolizable sugars, ligands of the recombinant protein etc.).
Cytoplasmic or periplasmic expression
As long as a cytoplasmic recombinant protein does not include stop-transfer sequences, the advantages of periplasmic secretion are also amenable to this type of protein. However, because stop-transfer sequences are difficult to predict, it is advisable to try both strategies in parallel. Using the vectors for N-terminal Strep-tag II or 6xHistidine-tag fusion, the change from cytoplasmic to periplasmic expression (and vice versa) can be achieved by a simple cloning step via the NheI/BstBI and the EcoRV/HindIII restriction sites on the 5'- and 3'- end, respectively (see Multiple Cloning Sites).
Do you need an authentic protein?
For special applications requiring an authentic protein, several vectors encoding the factor Xa protease cleavage site adjacent to Strep-tag II/6xHistidine-tag are available allowing the complete removal of the tag. In addition to factor Xa protease cleavage sites, we offer vectors with thrombin and enterokinase cleavage
sites. For an overview of all vectors click here. Please note, that in most cases it is superfluous to remove the tag.
| Factor Xa processing |
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Protein gel demonstrating the treatment of the 15 kDa selenoprotein (carrying a N-terminal Strep-tag) with factor Xa (1:1000, w/w) at 22°C. The gene encoding the protein had been cloned into pASK-IBA6, which contains a Xa cleavage site adjacent to Strep-tag II (see Multiple Cloning Sites ).
Lane 1, protein before Xa processing; lane 2, after 1 hour incubation; lane 3, after 2 hours; lane 4, after 4 hours; lane 5, after 16 hours: homogenous authentic protein without Strep-tag II. |
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