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Cloned gene in pET system not being expressed


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#1 marco.aso

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Posted 24 August 2015 - 09:34 AM

In order to access the function of modular domains of a multidomain protein I decided to amplify and clone separately the gene fragment that codes for each domain of this protein in pET28a vector, to posterior overexpression and purification of each protein domain. I divided the gene in 5 different fragments, all were digested with NdeI/BamHI (the restriction sites were added to the primers sequence), and cloned in pET28a digested with the same enzymes.

 

After cloning confirmation, the plasmids were transformed into BL21. Three of the proteins expressed normally, but two of them didn’t. Coincidence or not, these two genes were amplified with the same forward primer. I sequenced the inserts, and it looks fine, no frame problems or random mutagenesis. I don’t believe in a problem with codon usage, because the full-length protein expresses well in the same system. Someone have any suggestion about what is happening and what I could do to solve my expression problem?

 

Thanks in advance, 

 

Marco Oliveira. 



#2 phage434

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Posted 24 August 2015 - 10:03 AM

I assume you added a start codon to each fragment.



#3 marco.aso

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Posted 24 August 2015 - 10:05 AM

Yes, I did. Always the atg of the NdeI site (catatg) was used as start códon.

#4 marco.aso

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Posted 24 August 2015 - 10:10 AM

I am wondering if it could be caused by a stability problem. Does protein degration could be my problem?



#5 labtastic

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Posted 24 August 2015 - 02:29 PM

How are you checking for expression?  It may be possible two of your constructs are expressing in inclusion bodies. You'll see the expressed protein if you run whole cell's on SDS-PAGE, but you won't see it if you are purifying from soluble extract.

 

The other likely scenario is that those two isolated domains simply do not express. This happens all the time, especially when you appreciate the fact that your constructs are not "evolutionarily approved". If this is the case, you may consider modifying the boundary of your construct a little bit. Check secondary structure predictions around your construct boundaries and make you are not beginning or ending in the middle of a predicted secondary structure (i.e. make construct boundaries in unstructured loops whenever possible).

 

Another option is to express at 18C (I assume you're expressing at 37C?). 

 

You can also try expressing with a solubilizing tag if stability is an issue, i.e. SUMO, GFP, GST, etc.



#6 marco.aso

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Posted 24 August 2015 - 03:46 PM

Thank you very much for your help so far, phage and labtastic. 

 

I am checking the expression running the crude extract on SDS-PAGE, not trying to purify yet. The expression was performed at 30°C. 

 

I am also concerned about the truncation point that I chose. To define this point I tried to use secondary structure alignment. However, I realized that the results generated by distinct tools of aligment are very diverse, it is hard to ensure the lenght of each secondary structure. Which software with this purpose do you suggest me to use?


Edited by marco.aso, 24 August 2015 - 03:47 PM.


#7 labtastic

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Posted 25 August 2015 - 06:20 AM

How are you preparing your crude extract? Are you lysing, then centrifuging, then running the soluble (supernatant) fraction on the gel? Or are you solubilizing whole cells in SDS? I am trying to understand whether it is still possible your protein is actually expressing but into inclusion bodies. 

 

The reality is that secondary structure prediction softwares are only a best guess. No software is going to be exactly right, but it is slightly better than nothing, or randomly guessing. I'm sure whatever software you used is adequate.

 

In which case, consider making constructs with +/- 5 residues at each end. I once had to make 23 constructs of a particular protein until I got it to express.

 

And like I said before, try expressing with a cleavable solubilization tag. These can make a world of difference. You can get such fusion vectors from Addgene, or make your own using the pET28 backbone you have if you are comfortable with the cloning. They are simple to make, and would be a useful tool for the lab for decades to come.



#8 marco.aso

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Posted 26 August 2015 - 06:12 AM

I am lysing the whole cells by sonication, than I load at the SDS-PAGE this lysated, which I call crude extract. Hence, it includes the inclusion bodies. I also check the solubility of the protein: I transfer part of the crude extract to another tube, which I centrifuge. The supernatant is the soluble fraction, and the ressuspend pellet is the insoluble fraction, containing the inclusion bodies. Therefore, in my SDS-PAGE I analyze the crude extract, and the soluble and insoluble fractions. I am pretty sure that I am not losing the overexpressed protein somewhere during preparation of the sample. 

 

I think that it is a very good tip to change the truncation point. I will do it, hopefully it will work and in a couple of weeks I come back to tell the good news. And surely I will make my own set of fusion vectors, also a good idea.

 

Thank you very much indeed.



#9 labtastic

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Posted 26 August 2015 - 07:48 AM

Sounds like it's pretty clear it's not in inclusion bodies. That's just as well...purifying from IB's can be a real chore and should only be done when all other options have been exhausted.

 

Good luck making those new constructs. I suspect the effort to make and screen them will be worth it.



#10 marco.aso

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Posted 13 September 2015 - 07:33 AM

Hello colleagues,

 

I'm back to tell you the news. Actually, my expression issue is more interesting than the ones which we were speculating, and somehow is related to the labtastic hypotesis. The function of the protein that I chopped into domains is to modify a second protein. Turns out that some of the constructs were not being properly regulated, and they messed up the E. coli BL21 metabolism. Hence, these proteins were toxic to the cell. I had an insight that it could happen, and adding a specific nutrient to the culture would prevent the toxicity of the protein. Luckily, the two constructs that showed expression issues, were properly over-expressed on this condition. After many years of studying biology, it can still amaze me with little things. 

 

Cheers mates!


Edited by marco.aso, 13 September 2015 - 07:40 AM.


#11 labtastic

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Posted 14 September 2015 - 04:40 AM

Cool man, glad you figured out the issue.

 

Crazy how something small like adding an extra ingredient to the media can be the difference between nothing and everything. 

 

In grad school I had a similar issue...was growing a bug to purify out a native enzyme that required iron. If we supplemented the growth media with FeSO4, the bugs grew like crap and activity of the isolated protein was poor. If we supplemented growth media with Fe-EDTA instead, bugs grew 10x's better, the purified protein was highly active and preps were highly reproducible. Could have saved a year of my life had I known. 






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