Tet expression system

Features and benefits of the pASK-IBA vectors:

High-level expression in E. coli
Tightly regulated expression due to the tetracycline promoter
Enhanced stability of cytotoxic genes
Universal cloning strategy with one restriction enzyme
N- or C-terminal Strep-tag® II fusion
Cytosolic or periplasmic expression
Inexpensive induction with anhydrotetracycline
Ampicillin or chloramphenicol resistance

Principle and properties

pASK-IBA vectors work with the tightly regulated tetracycline (tet) promoter. Expression of the foreign gene is stringently repressed until efficient chemical induction with a low concentration of anhydrotetracycline. In contrast to the lac promoter- the tetA promoter/operator is tightly controlled and not functionally coupled to any cellular regulation mechanisms or genetic background. Therefore special E. coli strains or extra plasmids are not required as for the T7 promoter. The vectors do not mediate resistance against tetracycline.

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.

References

Tet promoter

Tet promoter:

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.
Degenkolb J, Takahashi M, Ellestad GA, Hillen W. (1991)
Structural requirements of tetracycline-Tet repressor interaction: Determination of equilibrium binding constants for tetracycline analogues with the Tet repressor.
Antimicrob. Agents Chemother. 35 (8), 1591-1595

pASK-Vectors

pASK-Vectors:

Müller I.B., Wu F., Bergmann B., Knöckel J., Walter R.D. (2009) Poisoning Pyridoxal 5-Phosphate-Dependent Enzymes: A New Strategy to Target the Malaria Parasite Plasmodium falciparum.PLoS ONE 4(2), e440.
Han, R., Zwiefka, A., Caswell, C.C., Xu, Y., Keene, D.R., Lukomska E., Zhao, Z., Höök, M., Lukomski, S. (2006) Assessment of prokaryotic collagen-like sequences derived from streptococcal Scl1 and Scl2 proteins as a source of recombinant GXY polymers. Appl. Microbiol. Biotechnol. 72, 109–115.
Humtsoe, J.O., Kim, J.K., Xu, Y., Keene, D.R., Höök, M., Lukomski, S. and Wary, K.K. (2005)
A streptococcal collagen-like protein interacts with the integrin and induces intracellular signalling.
JBC 280, 13848-13857.

pPR-IBA vectors

pPR-IBA vectors:

Eren E. Kennedy D.C., Maroney M. J., and Argüello J.M. (2006)
A Novel Regulatory Metal Binding Domain Is Present in the C Terminus of Arabidopsis Zn2 -ATPase HMA2,
JBC281 (45), 33881–33891

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