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Protein expression failing


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#1 Axel

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Posted 16 February 2010 - 04:46 AM

Ok, I'm getting desperate here. Sorry for the long text, there is a summary at the bottom. I just felt the need to write everything..

I'm doing my master's thesis at a company, and my job is to express a synthetic gene coding for a dehydrogenase that they hope to use in a process. It's a chemical company with a small biochem lab started about a year ago. The biochem branch of this project pretty much dies if I fail, and should I succeed I might get a job offer out of it. So far i'm failing, so I'm searching new input. I have sources that this specific protein has been expressed using a pTZ19R vector in DH5alpha E.coli cells, but apart from that, there are no details on how they did it.

What I've done:
Prepared pTZ19R vectors with the gene fragment by cutting out from the synthesis vector, ligating in and transforming. I've grown this in small cultures in 25, 30 and 37 degrees celcius over night, scaled up the culture and let grow for three hours, induced with 1 mM IPTG and then harvest the cells after 2 hours of expression. I've also tried different concentrations of IPTG while growing in 30 degrees.
This is then prepared and analyzed on SDS-PAGE. I use E.coli transformed with an empty pTZ19R vector as a negative control, otherwise treating it the same way.

No new bands show up on the gels, so I have tried a few different takes on the experiments:
*Removed 12 aa:s in the beginning of the gene, because the vector itself adds 12 aa before the actual ATG of the gene
*Moved the gene to pSE420, an expression vector already used by this lab.
*Sequencing showed that the gene was most likely missing a nucleotide, so I used PCR to insert the extra nucleotide in all four of the variants prevoiusly done (pTZ19R long & short, pSE420 long & short)

None of these tests show anything interesting, although I haven't tested at all temperatures for the latest ones. Additionally, I have screened for inclusion bodies using a protocol I found(http://structbio.van.../inclusion.html), stopping at step 15 (and using 8M Urea, not 8 mM) to run on SDS-PAGE. There is no difference between control and sample with gene here either.

My conclusion is that the expression seems to fail completely, I don't seem to get inclusion bodies and so far I see no real signs that any protein is manufactured at all. I mean to finish testing different temperatures for the latest clones and I'm waiting for new sequencing results. Though, from what I can tell the clones should be fine. I am thinking of prolonging the expression time to see if there is a difference. After tips from phage434 I will recommend a switch to BL21(DE3), but won't have the possibility to do so. But apart from that, I'm clueless. And judging from my results so far, I'm not hopeful to get a positive result before my time is up here.

So I turn to you, BioForum, for help. What could I change, where could I find the source of my failing experiments? All ideas are welcome, and I hope I can apply the better part of them. Thank you very much!

/Axel

Summary: My protein expression is failing, and I'm trying to find a solution by adjusting temperature and IPTG concentrations. Where else should I make changes?

#2 Vini

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Posted 17 February 2010 - 06:18 AM

Ok, I'm getting desperate here. Sorry for the long text, there is a summary at the bottom. I just felt the need to write everything..

I'm doing my master's thesis at a company, and my job is to express a synthetic gene coding for a dehydrogenase that they hope to use in a process. It's a chemical company with a small biochem lab started about a year ago. The biochem branch of this project pretty much dies if I fail, and should I succeed I might get a job offer out of it. So far i'm failing, so I'm searching new input. I have sources that this specific protein has been expressed using a pTZ19R vector in DH5alpha E.coli cells, but apart from that, there are no details on how they did it.

What I've done:
Prepared pTZ19R vectors with the gene fragment by cutting out from the synthesis vector, ligating in and transforming. I've grown this in small cultures in 25, 30 and 37 degrees celcius over night, scaled up the culture and let grow for three hours, induced with 1 mM IPTG and then harvest the cells after 2 hours of expression. I've also tried different concentrations of IPTG while growing in 30 degrees.
This is then prepared and analyzed on SDS-PAGE. I use E.coli transformed with an empty pTZ19R vector as a negative control, otherwise treating it the same way.

No new bands show up on the gels, so I have tried a few different takes on the experiments:
*Removed 12 aa:s in the beginning of the gene, because the vector itself adds 12 aa before the actual ATG of the gene
*Moved the gene to pSE420, an expression vector already used by this lab.
*Sequencing showed that the gene was most likely missing a nucleotide, so I used PCR to insert the extra nucleotide in all four of the variants prevoiusly done (pTZ19R long & short, pSE420 long & short)

None of these tests show anything interesting, although I haven't tested at all temperatures for the latest ones. Additionally, I have screened for inclusion bodies using a protocol I found(http://structbio.van.../inclusion.html), stopping at step 15 (and using 8M Urea, not 8 mM) to run on SDS-PAGE. There is no difference between control and sample with gene here either.

My conclusion is that the expression seems to fail completely, I don't seem to get inclusion bodies and so far I see no real signs that any protein is manufactured at all. I mean to finish testing different temperatures for the latest clones and I'm waiting for new sequencing results. Though, from what I can tell the clones should be fine. I am thinking of prolonging the expression time to see if there is a difference. After tips from phage434 I will recommend a switch to BL21(DE3), but won't have the possibility to do so. But apart from that, I'm clueless. And judging from my results so far, I'm not hopeful to get a positive result before my time is up here.

So I turn to you, BioForum, for help. What could I change, where could I find the source of my failing experiments? All ideas are welcome, and I hope I can apply the better part of them. Thank you very much!

/Axel

Summary: My protein expression is failing, and I'm trying to find a solution by adjusting temperature and IPTG concentrations. Where else should I make changes?



Hi Axel

sorry to hear aboutbthe problems you are facing.........see if these points are of any help....
1. I think you should also try increasing the time for which you keep ur induced culture. 2 hrs. appear to be way too less (i keep my induced cultures for at least 5 hrs).

2. Is this synthetic gene codon-optimized for expression in E.coli? If the gene shows GC/AT codon biasness, then u should resort to using RP/RIL competent cells respectively.

3. U can try uninduced cultures also. I know one colleague, whose protein used to come only with uninduced cultures.

4. try scaling up the culture to 2-4 lt.

5. try a lower temperature...... 22, 18 degrees.


all the best!




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