Champion™ pET SUMO Expression System
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Champion™ pET SUMO Expression System

The Champion™ pET SUMO Expression System produces the highest levels of soluble protein in E. coli. It utilizes a smallRead more
Catalog NumberQuantity
K3000120 reactions
Catalog number K30001
Price (JPY)
208,900
Each
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Quantity:
20 reactions
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The Champion™ pET SUMO Expression System produces the highest levels of soluble protein in E. coli. It utilizes a small ubiquitin-related modifier (SUMO) fusion, belonging to the growing family of ubiquitin-related proteins, to enhance the solubility of expressed fusion proteins. In contrast to ubiquitin, SUMO is involved in the stabilization and localization of proteins in vivo. After expression, the 11 kd SUMO moiety can be cleaved by the highly specific and active SUMO (ULP-1) protease at the carboxyl terminal, producing a native protein*. The Champion™ pET SUMO Protein and Peptide Expression System offers:

• Greatly enhanced solubility with an N-terminal SUMO fusionHighly efficient cleavage- produces native protein of interest with SUMO (ULP-1) protease*Highly specific cleavage- eliminates the chance of your protein of interest being internally digested, regardless of its amino acid sequenceSignificantly increased stability with SUMO fusion-can be used for small peptide productionT7lac promoter for high-level protein expressionN-terminal 6xHis tag for protein detection and purification
For Research Use Only. Not for use in diagnostic procedures.
Specifications
Antibiotic Resistance BacterialKanamycin (KanR)
Bacterial or Yeast StrainBL21(DE3)
CleavageSUMO
Constitutive or Inducible SystemInducible
Expression MechanismCell-Based Expression
Expression SystemE. coli
Inducing AgentIPTG
Product TypeExpression System
Quantity20 reactions
Selection Agent (Eukaryotic)None
VectorpET
Cloning MethodTOPO
Product LineChampion™, TA Cloning™
PromoterT7, lacO
Protein TagHis Tag (6x), SUMO Tag
Unit SizeEach
Contents & Storage
The Champion™ pET SUMO Expression System is provided as a complete system. The Champion™ pET SUMO TA Cloning™ box contains linearized Champion™ pET SUMO vector, sterile water, dNTPs, 10X PCR Buffer, control template and primers, T4 DNA ligase, 10X ligation buffer, primers for sequencing or PCR screening, and an expression control. Store at -20°C. The SUMO Protease box contains SUMO Protease and buffers. Store at -80°C. The One Shot™ TOP10 box contains twenty-one 50 μl aliquots of chemically competent E. coli, S.O.C. medium, and a control plasmid. Store at -80°C. The One Shot¤ BL21(DE3) box contains twenty-one 50 μl aliquots of chemically competent E. coli, S.O.C. medium, and a control plasmid. Store at -80°C.
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Frequently asked questions (FAQs)

My gene of interest is toxic to bacterial cells. Are there any precautions you can suggest?

Several precautions may be taken to prevent problems resulting from basal level expression of a toxic gene of interest. These methods all assume that the T7-based or Champion-based expression plasmid has been correctly designed and created.

- Propagate and maintain your expression plasmid in a strain that does not contain T7 RNA polymerase (i.e., DH5α).
- If using BL21 (DE3) cells, try growing cells at room temperature rather than 37 degrees C for 24-48 hr.
- Perform a fresh transformation using a tightly regulated E. coli strain, such as BL21-AI cells.
- After following the transformation protocol, plate the transformation reaction on LB plates containing 100 µg/mL ampicillin and 0.1% glucose. The presence of glucose represses basal expression of T7 RNA polymerase.
- Following transformation of BL21-AI cells, pick 3 or 4 transformants and inoculate directly into fresh LB medium containing 100 µg/mL ampicillin or 50 µg/mL carbenicillin (and 0.1% glucose, if desired). When the culture reaches an OD600 of 0.4, induce expression of the recombinant protein by adding L-arabinose to a final concentration of 0.2%.
- When performing expression experiments, supplement the growth medium with 0.1% glucose in addition to 0.2% arabinose.
- Try a regulated bacterial expression system such as our pBAD system.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I'm trying to express my protein using a bacterial expression system. How do I know if I'm seeing degradation of my protein or if what I’m seeing is codon usage bias?

Typically, if you see 1-2 dominant bands, translation stopped prematurely due to codon usage bias. With degradation, you usually see a ladder of bands. With degradation, you can try using a protease inhibitor and add it to the lysis buffer to help prevent degradation. If degradation is the issue, a time point experiment can be done to determine the best time to harvest the cells.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I'm trying to express my protein using a bacterial expression system and am getting inclusion bodies. What should I do?

If you are having a solubility issue, try to decrease the temperature or decrease the amount of IPTG used for induction. You can also try a different, more stringent cell strain for expression. Adding 1% glucose to the bacterial culture medium during expression can also help.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I'm getting low protein yield from my bacterial expression system. What can I do to improve this?

- Inoculate from fresh bacterial cultures, since higher protein yields are generally obtained from a fresh bacterial colony.

- Check the codon usage in the recombinant protein sequence for infrequently used codons. Replacing the rare codons with more commonly used codons can significantly increase expression levels. For example, the arginine codons AGG and AGA are used infrequently by E. coli, so the level of tRNAs for these codons is low.

- Add protease inhibitors, such as PMSF, to buffers during protein purification. Use freshly made PMSF, since PMSF loses effectiveness within 30 min of dilution into an aqueous solution.

- If you are using ampicillin for selection in your expression experiments, you may be experiencing plasmid instability due to the absence of selective conditions. This occurs as the ampicillin is destroyed by β-lactamase or hydrolyzed under the acidic media conditions generated by bacterial metabolism. You may want to substitute carbenicillin for ampicillin in your transformation and expression experiments.

- The recombinant protein may be toxic to bacterial cells. Try a tighter regulation system for competent cell expression such as BL21-AI. You may also consider trying a different expression system such as the pBAD system.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

My cells are growing very slowly, and I'm not getting any protein expression from my baterial expression system. What can I do to fix this?

This typically occurs when your gene of interest is toxic. Try using a tighter regulation system, such as BL21 (DE3) (pLysS) or BL21 (DE3) (pLysE), or BL21(AI).

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

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Vector Information

Vector Name
Vector Map
Polylinker
Sequence
Restriction
pIND-E/Uni/lacZ
pET SUMO/CAT

Citations & References (1)

Citations & References
Abstract
A novel hematopoietic granulin induces proliferation of goldfish (Carassius auratus L.) macrophages.
Authors:Hanington PC, Barreda DR, Belosevic M,
Journal:J Biol Chem
PubMed ID:16473876
'Granulins are a group of highly conserved growth factors that have been described from a variety of organisms spanning the metazoa. In this study, goldfish granulin was one of the most commonly identified transcripts in the differential cross-screening of macrophage cDNA libraries and was preferentially expressed in proliferating macrophages. Unlike ... More
1 total citations

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