Protein didn't express - why? - M15 E. coli culture and pQE30UA vector; pTrcHis2TOPO and TOP10 E. coli cells (Jun/18/2006 )

Hi everyone,

Please help! I have ligated my gene into the pTrcHis2TOPO vector (transformed TOP10 E. coli) and the Qiagen pQE30UA vector (transformed M15 cells). PCR analysis has confirmed the presence of the gene in the transformed cells, BUT no protein appears to be expressed from my inductions.

Here is the protocol that I have been using:

Resuspend colony in 3microL water (use one for PCR analysis)
Transfer remaining 2microL into 10ml LB media (M15: add 10microL of ampicillin (100mg/ml) and 5microL kanamycin (50mg/ml stock); TOP10: add 5microL ampicillin and glucose to 0.5%. Incubate overnight at 37 with shaking.

Next morning: add the 10ml of culture to 40ml LB media (my supervisor told me that I do not need to add antibiotics at this point because of the high concentration of cells). For the TOP10 cells I add glucose again to 0.5%. Incubate with shaking, 37 degrees.

After 4 hours, check the OD. I know that OD is supposed to be over 0.5 or 0.6, but I have been using a 96 well plate (200microL sample) to read OD which I have been told is not accurate because the light is not passing through a 1cm cuvette (definition of OD). Anyway, my OD through that has been around 0.4, which other users in my lab say is ok. For the TOp10 cells, I spin down the cells and resuspend in glucose free media.

Then, I have been adding 150microL of 450mM IPTG. Adding this to a 50ml culture gives an (approximately) 1.5mM final concentration???

Then I put back in incubator with shaking and take samples at 0hrs, 2hrs and 3hrs. My supervisor told me that 3hrs is generally the optimum expression time.

Have I been doing something wrong? What can you suggest I try? Thanks for reading my long post

I am a student and I am getting very disheartened because everything seems to go wrong, and I feel like my supervisors must think I am not a very good student

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have you sequenced your construct?

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Sequencing is underway at the moment, I have tried a few different concentrations for sequencing pcr but so far the results have come back very poor, so i have not been able to interpret them. Construct amplifies successfully by PCR though, with primers that are specific to the 5' and 3' ends of the gene

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have you tried PCR w/ primers specific for the plasmid that flanks the insert? where did your gene come from, if it came from e. coli, then your primers might be amplifying the genome (top10 or M15 cells) instead of your insert. Also as stated above, sequencing is a must IMO to check for PCR mutations in your insert.

Also sequencing, are u sequencing the plasmid itself or PCR products?

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Hi,

I have amplified the colonies successfully with both vector specific primers and gene specific primers. PCR using the flanking primers showed an insert of the right size, and PCR using gene specific showed a single strong band at the correct size for the gene also. DIgestion of the plasmid yields a fragment the same size as my insert.

My gene is a human one that is involved in cell signalling, and is not present in E. coli.

I have just found a method to quantify the amount of DNA in my vector prep by spotting onto agar and comparing to standards (doing this now), so should have the sequences back in a few days. I am sequencing the plasmid, using a forward primer that is specific to the pQE30UA vector and will probably set up another one using the reverse primer. The problem with the pTrcHis2TOPO vector is that I do not have any vector specific primers, so I will be using gene specific ones.

HOw often do PCR's mutate? I have used different PCR products to ligate into each vector because for one of the vectors I had to add a His-tag to the start of the gene. Would it be unusual for two different PCRs to mutate?

Is it possible that the protein is so toxic to the E. coli that it is being degraded / the cells are dying and that is why it is not seen at all in the SDS-PAGE?

Thanks for your help everyone

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here is the sequence of your vector (the one with the trc promoter)

this is the sequence of your polylinker


if I were you, I would make some primers that correspond to flanking vector sequence, and use them to sequence that clone. It is possible that you may have a toxic protein; it happens and there are things you can do to reduce expression in order to get purified protein, usually

however, before spending weeks and weeks attempting to optimize your protein prep, I would be 100% sure that your construct is what you think it is. polymerases do make errors, and so do we in designing primers...you may have a resulting frameshift mutation at one end or the other that is at fault in your situation.


let us know what happens?

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Hi:
Firstly: you must confirm whether the construct is right.
Could you give us some details:
1. MW
2. His tag?
3. what method do you choose to detect the protein expression? (inclusion body or supernatant)
4. IS the protein prone to degradation?
5. How many strains do you pick to do pilot expression? (Normally choose more than 1)
6. What is the antibiotic concentration in indution medium? (If the plasmid is prone to lost, you may add more antibiotic)
Believe youself!

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Thanks so much for your help everyone. I had one of the vectors sequenced and the gene is in the wrong way! I didn't realise that my primers would amplify the fragment if it was in the wrong way... *blushes*

I am sequencing the other one now, but I can only assume that the same thing has happened with that one.



At least I have identified the problem and it can be semi-easily fixed...

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If you do a PCR with one of your primers in the gene of interest and the other out in the vector then it would be easy to see that the insert was in the wrong orientation based on size of the product.

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Does your gene of interest contain the start codon?
Sometimes, the protein is expressed but the antibody can't detect
Check if the antibody is working using the positive control first

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