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Product FAQ

I'm getting no expression from my bacterial expression vector, but my cells are growing normally. What should I do?

Answer

Please view the possible causes and solutions to try:

- Frame shifts or a premature stop codon is present in the construct; check the sequence.
- The wrong cell strain was used for expression.
- If using a glycerol stock, the integrity of the plasmid can change because most cell strains for expression are not RecA and EndA-. Use freshly transformed cells.
- The protein is in the insoluble section; check cell lysates and not just the supernatant.
- Rare codons were used in the gene of interest: check the codon usage. (http://nihserver.mbi.ucla.edu/RACC)
- The cells may be kicking out the plasmid during culture: this is more common in plasmids that are ampicillin resistant. Try using carbenicillin instead of ampicillin in the medium; wash and resuspend the overnight culture with LB containing fresh amp/carb before inoculation.

Answer Id: E9736

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Product FAQ

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?

Answer

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).

Answer Id: E9737

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Product FAQ

What factors can affect expression in an inducible T7 system?

Answer

There are several factors that can affect expression including:

- Amount of inducer (IPTG) added
- Time of induction (optimal OD600 for induction is 0.4 to 0.6)
- Duration of induction
- Induction temperature
- The construct itself

Answer Id: E9701

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Product FAQ

What is the optimal spacing between the ribosome-binding site (RBS) and the ATG when cloning into a bacterial expression vector?

Answer

The sequence between the RBS and ATG should be between 8-12 bp and not contain any palindromic sequence.

Answer Id: E9696

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Product FAQ

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

Answer

- 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.

Answer Id: E9738

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Product FAQ

Can I use one of your mammalian expression vectors with a T7 promoter for expression in E. coli?

Answer

Transcripts can be made, but there is no ribosome binding site or Shine Dalgarno sequence to initiate translation; therefore, little protein will be produced.

Answer Id: E9702

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Product FAQ

How much IPTG can I use to induce expression from a T7 promoter containing bacterial expression vector?

Answer

This can vary somewhat, but we typically suggest a starting range of 0.1-5 mM IPTG.

Answer Id: E9697

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Product FAQ

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

Answer

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.

Answer Id: E9739

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Product FAQ

Are NP40 and DTT required for SUMO™ Protease activity? Will they interfere with downstream applications?

Answer

Yes, they are both required, although it might be possible to substitute the NP40 with another detergent via dialysis if you have concern about interference with mass spectrometry. DTT is an absolute requirement.

Answer Id: E4965

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Product FAQ

What is the yield I should expect from my T7 promoter-based expression system?

Answer

The T7 promoter-based expression systems usually give fairly high yield and can be scaled up easily. Yields will vary depending on the protein being expressed, but in general yields range from 100 μg to 10 mg per liter of culture.

Answer Id: E9703

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Product FAQ

Can I co-express two proteins in E. coli?

Answer

To express two proteins at the same time in E. coli, we suggest using a dual promoter vector or using two different but compatible vectors at the same time. For example, you could try a pET vector with a pRSET vector, which contain different ORI (pBR322 origin and pUC origin, respectively). The only issue is that the pRSET vector is high copy number but pET is not; therefore, you may get significantly more protein expression from pRSET than from pET if you add them into one host cell.

Answer Id: E9694

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Product FAQ

I am using a bacterial expression vector containing the T7 promoter. At what OD should I induce my cells?

Answer

The time of induction can vary widely. Successful experiments using the T7 systems have induced at an OD600 of 0.1-1.2. Generally speaking, induction at high ODs will lead to lower expression yields, as the cells will stop growing rapidly after the density is too high. The optimal OD600 for induction is 0.4 to 0.6.

Answer Id: E9698

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Product FAQ

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?

Answer

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 this is the case, a time point experiment can be done to determine the best time to harvest the cells.

Answer Id: E9740

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Product FAQ

Can I use carbenicillin in place of ampicillin in my transformation/T7 expression experiments?

Answer

Yes; in fact, carbenicillin is generally more stable than ampicillin and may help to increase expression levels by preventing loss of the pET plasmids.

Answer Id: E9704

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Product FAQ

What protein yields should I expect to get with the pRSET expression vectors?

Answer

We've expressed CAT and Beta-Gal in volumes from 2 ml to 50 ml and get about 20-50 µg protein/ml of culture. Protein yield is dependent on the type of protein being expressed and the culturing conditions.

Answer Id: E3259

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