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cis-Repressed pQE Kan Vector Set

Cat. No. / ID: 32943

25 ug each: pQE-80-L-Kan, pQE-81L-Kan, pQE-82L-Kan

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Product Details

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Specifications

FeaturesSpecifications
All three reading frames providedYes
Tag6xHis tag
Antibiotic resistancecis-Repressed pQE Vector Set: Ampicillin; cis-Repressed pQE KAN Vector Set: Kanamycin
Expression speciesE. coli
N- or C-terminal tagN-terminal tag
ExpressionIn vivo
Special featuresAdditional expression of the laclq gene
In-frame cloning necessaryYes
Tag removal sequenceNo

Resources

Selection Guides (1)
Kit Handbooks (1)
A handbook for high-level expression and purification of 6xHis-tagged proteins
Supplementary Protocols (1)
For pQE-80L, pQE-81L, and pQE-82L vectors

FAQ

How can I express toxic protein in E. coli?

To reduce the effects of protein toxicity on bacterial cell growth prior to induction, the level of basal transcription that occurs in the absence of induction (“leakiness”) should be repressed as much as possible, and the number of generations before induction should be kept to a minimum.

For very toxic proteins, we recommend using the pQE-80L series of expression vectors in the M15[pREP4] E. coli host strain. The pQE-80L vectors have a cis-lacIq gene that overexpresses the lac repressor, in addition to a lacI repressor gene present in trans on a separate pREP4 plasmid. This combination of two repressor modules results in highly efficient suppression of recombinant protein expression prior to induction and gives the best chance of successful expression of toxic proteins. Furthermore, induction of the toxic protein should be done at a time point as late as possible, and the IPTG concentration used for induction should be reduced. Conditions have to be optimized for each application.

Another alternative is to use cell-free protein biosynthesis systems , like the EasyXpress Protein Synthesis Kit. The advantages of cell-free expression systems include time savings, the possibility to produce proteins that are toxic and the ability to adapt reaction conditions to the requirements of the synthesized protein.

 

FAQ ID -373
What is the origin of replication and the plasmid copy number of the pQE vectors?
The QIAexpress pQE vectors contain a pBR322 derived ColE1 origin of replication and are classified as low-copy plasmids (by our estimate, approximately 20-30 copies per cell; exact numbers have not been determined). The pQE-TriSystem Vector has a pUC origin of replication and is classified as a high-copy vector. Please see also FAQ 350 for general information on replication origins and copy numbers of various commonly used plasmids.
FAQ ID -338
How should I propagate pQE expression plasmids?

QIAexpress pQE vectors and constructs can be maintained in any E. coli strain that is ampicillin-sensitive and carries the pREP4 repressor plasmid, or harbors the lacIq gene on the F-factor episome.

M15 and SG13009 E. coli host strains carry lacI on the plasmid pREP4, while XL1-Blue or the JM series contain an episomal copy of lacIq.

LacIq is a mutation of lacI that produces very high levels of the lac repressor. Initial cloning and propagation using XL1-Blue is recommended because plasmid preparations derived from QIAexpress host strains will also contain pREP4 DNA, which could make clone analysis more difficult.

Alternatively, the pQE-80L series of expression vectors which encodes a lacIq repression module, allows use of any E. coli host strain.

FAQ ID -58
How can I be sure that I am harvesting my induced bacterial culture at the best time point for protein expression?

To optimize the expression of a given recombinant protein, a time-course analysis of the level of protein expression in the induced culture is recommended. Intracellular protein content is often a balance between the amount of soluble protein in the cells, the formation of inclusion bodies, and protein degradation. By checking the 6xHis-tagged protein present at various times after induction in the soluble and insoluble fractions, the optimal induction period can be established, and the bacterial culture can be harvested at this time. It may be useful to perform plasmid Mini preparations on culture samples during the time-course to enable monitoring of plasmid (expression construct) maintenance.

Below, you can see an example of a time course of recombinant protein expression using the QIAexpress System. You can find this information also in the Section 'Expression in E. coli' in the QIAexpressionist Handbook. The handbook is an important resource for useful background information and protocols. For instructions on how to isolate protein from the soluble and insoluble fractions of induced cultures please see Protocol 14. "Protein minipreps of 6x His-tagged proteins from E. coli under native conditions" and Protocol 19. "6xHis-tagged protein minipreps under denaturing conditions."

 

 

 

Time course of expression using the QIAexpress System. Expression of 6xHis-tagged DHFR was induced with 1 mM IPTG. Aliquots were removed at the times indicated and purified on Ni-NTA Agarose under denaturing conditions. Proteins were visualized by Coomassie staining. Yields per liter culture were 2.8, 5.5,12.3, 33.8, and 53.9 mg, respectively. ■A Crude cell lysate; ■B purification with Ni-NTA. 1: flow-through, 2 & 3: first and second eluates; M: markers; C: noninduced control.

 

 

FAQ ID -788