S_1125_2_C_Terminus_pQE_Vector_Set

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C-Terminus pQE Vector Set

Cat. No. / ID:  32903

pQE-16, pQE-60, pQE-70 각각 25µg
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₩1,141,000.00
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C-Terminus pQE Vector Set은/는 분자생물학 분야에 사용하기 위한 것입니다. 이 제품은 질병의 진단, 예방, 또는 치료용이 아닙니다.

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Features

  • C 말단 6xHis 태그가 있으면 전장 단백질만 정제됩니다.
  • DHFR 융합체로서 저조하게 발현된 단백질의 발현을 위한 pQE-16 벡터
  • DHFR 융합체로서 짧은 펩티드의 발현을 위한 pQE-16 벡터

Product Details

이 세트는 C 말단 6xHis 태그 단백질 발현을 위한 세 가지 벡터(pQE-16, pQE-60, pQE-70)를 제공하며, 조기 종료를 유발할 수 있는 ‘일시 중지 부위’가 있는 개방형 판독 프레임에 사용하는 것이 좋습니다. pQE-60 및 pQE-70을 사용하면 코딩 단편의 원래 시작 코돈이 pQE 벡터의 ATG를 대체하여 단백질의 실제 N 말단을 보존할 수 있습니다. 이러한 두 구축물은 PCR 또는 돌연변이 유발에 의해 삽입물의 ATG 코돈에 각각 NcoI 및 SphI 제한 부위를 주입하여 생성합니다. pQE-16을 사용하면 C 말단 6xHis 태그 DHFR 융합 단백질이 발현됩니다. 디히드로엽산 환원효소(Dihydrofolate Reductase, DHFR)은 항원성과 안정성을 강화하며, 발현이 저조한 단백질이나 단백질이 분해되기 쉬운 짧은 펩티드에 사용하는 것이 좋습니다. DHFR 자체는 마우스와 rat에서 면역원성을 거의 보이지 않기 때문에 DHFR 융합 단백질은 에피토프 스크리닝에 적합합니다.

Principle

QIAexpress pQE 벡터는 강력한 파지 T5 프로모터(E. coli RNA 중합효소로 인식)와 이중 유당 작동 유전자 억제 모듈을 결합하여 E. coli에서 엄격하게 조절되는 높은 수준의 재조합 단백질 발현을 제공합니다(그림 ' QIAexpress pQE 벡터' 참조). 높은 수준의 유당 억제인자가 있는 경우 단백질 합성이 효과적으로 차단되고 세포독성 구축물의 안정성이 향상됩니다.  pQE 벡터를 사용하면 재조합 단백질의 N 또는 C 말단에 6xHis 태그를 배치할 수 있습니다.

 

QIAexpress pQE 벡터에 포함된 요소
구성 요소 설명
최적화된 프로모터/작동 유전자 요소 파지 T5 프로모터와 유당 작동 유전자 염기서열 두 개로 구성되며, 이 염기서열은 lac 억제자 결합 가능성을 높이고 강력한 T5 프로모터를 효율적으로 억제합니다.
합성 리보솜 결합 부위 RBSII 효율적인 번역에 사용
6xHis 태그 코딩 염기서열 폴리링커(polylinker) 복제 영역의 5' 또는 3'
번역 중지 코돈 발현 구축물을 편리하게 준비할 수 있도록 모든 판독 프레임에서
강력한 전사 종결자 2개 파지 람다의 t0E. coli rrnB 오페론의 T1, 연속 판독 전사를 방지하고 발현 구축물의 안정성 보장
ColE1 복제 기점 pBR322에서
Beta-lactamase gene (bla) 암피실린 내성 부여
See figures

Procedure

관심 단백질을 인코딩하는 삽입물이 적절한 구조로 복제되고(자세한 정보는 QIAexpressionist 안내서 참조) 발현에 적합한 E. coli 균주로 변환됩니다. 발현은 IPTG를 첨가하면 유도됩니다. 벡터 pQE-TriSystem 구축물은 E. coli로 변환되어 곤충 세포에서 재조합 단백질 발현을 위한 셔틀 벡터로 사용되거나 포유류 세포로 변환됩니다.

Applications

QIAexpress Expression System은 다음을 포함하여 다양한 응용 분야에 적합한 높은 수준의 단백질 발현을
제공합니다.

  • 기능 및 구조적으로 활성 상태인 단백질의 정제
  • 항체 생성을 위한 변성 조건에서의 정제
  • 3차원 구조 측정을 위한 결정화
  • 단백질-단백질 및 단백질-DNA 상호 작용을 포함하는 분석

Supporting data and figures

Specifications

FeaturesSpecifications
Expression speciesE. coli
In-frame cloning necessary
N- or C-terminal tagC-terminal tag
ExpressionIn vivo
Special featuresT5 프로모터 전사-번역 시스템 기반
Tag6xHis tag
Tag removal sequence아니요
All three reading frames provided

Resources

Selection Guides (1)
Vector Sequences & Maps (3)
For the pQE-16 vector
For the pQE-60 vector
For the pQE-70 vector
Safety Data Sheets (1)
Kit Handbooks (1)
A handbook for high-level expression and purification of 6xHis-tagged proteins
Certificates of Analysis (1)

FAQ

Why is the pQE DNA provided in QIAexpress Kits blue in color?
The blue color results from bromophenol blue added to the plasmid DNA prior to lyophilization to make the DNA more easily visible. After the DNA pellet is resuspended in water or Tris-HCl, the dye will not inhibit downstream applications such as transformation of bacterial cells or restriction enzyme digestion.
FAQ ID -487
What is the size, charge and isoelectric point of the DHFR protein in the pQE vectors?

The native Dihydrofolate Reductase (DHFR) protein has the following technical features:

  • Molecular Weight: 21.5 kDa
  • Isoelectric Point: 8.5
  • Charge at pH 7.0: 2.2

DHFR expressed in QIAexpress vectors pQE-16 and pOE-40 has the following technical features:

pQE-16

  • Molecular Weight: 23.1 kDa
  • Isoelectric Point: 9.1
  • Charge at pH 7.0: 5.2

pQE-40

  • Molecular Weight: 24.5 kDa
  • Isoelectric Point: 9.2
  • Charge at pH 7.0: 7.2
FAQ ID -470
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
Which primers can I use for sequencing pQE-expression vector constructs?
All pQE vectors (except pQE-TriSystem) can be sequenced using any of the primers described on page 118 of the QIAexpressionist.
FAQ ID -343
Is dihydrofolate reductase (DHFR) immunogenic?

Murine DHFR, used as a fusion protein to enable expression of very small proteins and peptides with the QIAexpress System, is poorly immunogenic in mouse and rats. It may however lead to an immune response in rabbits and goat. DHFR protein is encoded in the QIAexpress vectors pQE-16 or pQE-40.

FAQ ID -471
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