pQE-TriSystem Vector

단일 구조를 사용하여 E. coli, 포유류 세포, 배큘로바이러스에 감염된 곤충 세포에서 His-tagged 단백질의 병렬 발현에 사용

S_1126_4_pQE_TriSystem_Vector

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pQE-TriSystem Vector

Cat. No. / ID:  33903

25µg pQE-TriSystem Vector DNA
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pQE-TriSystem Vector은/는 분자생물학 분야에 사용하기 위한 것입니다. 이 제품은 질병의 진단, 예방, 또는 치료용이 아닙니다.

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✓ Knowledgeable and professional Product & Technical Support

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Features

  • 시간이 오래 걸리는 서브클로닝 절차가 필요 없음
  • 곤충 또는 포유류 세포에서 번역 후 변형 획득
  • 하나의 구조체로 세 가지 발현 시스템에서 효율적인 발현 제공

Product Details

pQE TriSystem Vector를 사용하면 세 가지 다른 발현 시스템을 포함하는 단일 벡터에서 His-tagged 단백질을 높은 수준으로 발현시킬 수 있습니다. E. coli,에서 발현하기 위한 T5 promoter/lac operator 전사 번역 시스템, 곤충 세포에서 배쿨로바이러스 기반 발현을 위한 p10 promoter, 포유류 세포에서 발현하기 위한 CAG(CMV/액틴/글로빈) promoter가 있습니다.

Performance

pQE-TriSystem Vector에는 포유류, E. coli, 배쿨로바이러스에 감염된 곤충 세포에서 각각 6xHis-tagged 단백질 발현을 가능하게 하는 CAG, T5, p10 promoter가 포함되어 있습니다(그림  pQE TriSystem 참조). E. coli 중합효소에 의해 인식되는 강력한 T5 promoter를 사용하여 박테리아 발현 시스템에서 예비 연구를 수행할 수 있으며, 모든 E. coli 균주에서 단백질을 효율적으로 발현할 수 있습니다. 예를 들어 번역 후 변형을 획득하기 위해 포유류 또는 곤충 세포에서의 발현이 필요한 경우, 시간이 많이 걸리는 서브클로닝 절차 없이도 동일한 구조를 사용할 수 있습니다.
See figures

Principle

QIAexpress pQE 벡터는 강력한 파지 T5 promoter(E. coli RNA 중합효소에 의해 인식됨)와 이중 lac operator 억제 모듈을 결합하여 E. coli에서 엄격하게 조절된 고수준의 재조합 단백질 발현을 제공합니다. 고수준의 lacrepressor가 존재하면 단백질 합성이 효과적으로 차단되고 세포 독성 구조체의 안정성이 향상됩니다. pQE 벡터( pQE Vector 표 및 그림 참조)를 사용하면 재조합 단백질의 N-말단 또는 C-말단에 6xHis tag를 넣을 수 있습니다.

QIAexpress pQE Vector에 존재하는 구성 요소
구성 요소 설명
1. 최적화된 promoter/operator 구성 요소

파지 T5 promoter와 두 개의 lac operator 염기서열로 구성됩니다

이는 lac repressor의 결합 가능성을 높이고

강력한 T5 promoter의 효율적인 억제를 보장합니다

2. 합성 리보솜 결합 부위 RBSII 효율적인 번역을 위해 사용
3. His-tag 코딩 염기서열 5' 또는 3'에서 polylinker 클로닝 영역까지
4. 번역 중지 코돈 모든 판독 프레임에서 발현 구조체를 편리하게 준비할 수 있습니다
5. 두 가지 강력한 전사 종결 인자

파지 람다의 t0, E. coli rrnB 오페론의 T1

연속 판독(read-through) 전사를 방지하고 발현 구조체의 안정성을 보장

6. ColE1 복제 기점

pBR322 유래
7. 베타 락타마제 유전자(bla) 암피실린 내성 부여
See figures

Procedure

관심 단백질을 코딩하는 삽입물을 적절한 구조체로 복제하고 발현에 적합한 E. coli 균주로 변형합니다. IPTG를 추가하면 발현이 유도됩니다. 벡터 pQE-TriSystem 구조체는 E. coli로 형질 전환하거나, 곤충 세포에서 재조합 단백질 발현을 위한 셔틀 벡터로 사용하거나, 포유류 세포에 감염시킬 수 있습니다.

Applications

QIAexpress Expression System은 다음을 포함한
많은 애플리케이션에 적합한 고수준 단백질 발현을 제공합니다.

  • 기능적, 형태적으로 활성화된 단백질의 정제
  • 항체 생산을 위한 변성 조건에서의 정제
  • 3차원 구조 결정을 위한 결정화
  • 단백질-단백질 및 단백질-DNA 상호작용 관련 분석

Supporting data and figures

Specifications

FeaturesSpecifications
In-frame cloning necessary
Expression체내
Tag removal sequence아니요
Expression speciesE.coli, 포유류 및 곤충 세포
Tag6xHis tag
N- or C-terminal tagC-말단 태그
All three reading frames provided아니요

Resources

Safety Data Sheets (1)
Selection Guides (1)
Kit Handbooks (1)
A handbook for high-level expression and purification of 6xHis-tagged proteins
Certificates of Analysis (1)

FAQ

Is it possible to use QIAGEN's pQE-TriSystem Vectors with the EasyXpress Protein Synthesis Insect Kit?

No, the pQE-TriSystem vectors contain a T5 Promoter. The expression plasmid used with the EasyXpress Protein Synthesis Insect Kit must contain a T7 Promoter.

FAQ ID -876
How can I increase expression of my 6xHis-tagged protein in E. coli?

Low-level expression can occur because the protein is toxic or unstable, or because the expression construct is not maintained in the cells during growth. In some cases, the 5' end of the inserted DNA sequence may encode elements that interfere with transcription or translation (e.g., masking of the Shine-Dalgarno sequence by stem-loop structures resulting from inverted repeats). In these instances, the sequence being expressed should be checked and modified if necessary. Modifications of growth media and different host strains may also have an effect on expression.

Please review the section "Specific considerations" in the Chapter 'Expression in E. coli' of the QIAexpressionist Handbook and refer to standard literature in protein science (e.g., Current Protocols in Protein Science, eds. John Wiley and Sons, New York) for additional information.

FAQ ID -63
How can I increase the amount of soluble recombinant protein in E. coli expression?

The QIAexpress Protein Purification System allows easy solubilization of 6xHis-tagged proteins sequestered into insoluble inclusion bodies by using denaturants such as 6 M Guanidine-HCl or 8 M Urea, or a variety of detergents. Proteins purified under denaturing conditions can then be refolded if necessary before use (please see: Wingfield, P. T., Palmer, I., and Liang, S.-M. (1995). Folding and purification of insoluble (inclusion-body) proteins from Escherichia coli. In: Current Protocols in Protein Science, vol. 1, Coligan, J. E., Dunn, B. M., Ploegh, H. L., Speicher, D. W., and Wingfield, P.T. eds. Wiley and Sons, Inc. New York, pp. 6.5.1–6.5.27.). The QIAexpressionist also contains recommendations for refolding proteins prior to the Appendix section of the handbook.

To increase levels of soluble protein, here are a few recommendations:

  • a reduction in growth temperature following induction may be helpful. Growth temperature often directly affects both expression levels and protein solubility, and lower temperatures will reduce expression levels leading to a higher amount of soluble protein.
  • the culture can be grown to a higher cell density before induction and the expression period can be kept to a minimum.
  • The IPTG concentration can be reduced from 1 mM to 0.005 mM, which would reduce the expression level by 90–95%.
  • it may be sufficient to change the host strain used, since certain strains tolerate some proteins better than others and allow higher levels of expression before forming inclusion bodies.
  • many proteins require metal cofactors in order to remain soluble, and the addition of metal salts to the culture media may be helpful. If the metal requirements of the protein are not known, a number of different supplements should be tested. Note that some divalent cations may interfere with protein binding to Ni-NTA.

 

 

FAQ ID -64
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
Do pQE vectors contain the CAT gene?
The chloramphenicol acetyl transferase gene (CAT) present between t0 and T1 has no promoter and is not normally expressed. Depending on the bacterial strain and insert,low CAT activities may be detectable
FAQ ID -362
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