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expreesing cloned vector


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#1 floxed

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Posted 05 July 2011 - 01:18 PM

Hi.
I have been trying to make 3 deletion mutants of a 2.2kb gene. the deletions are in the center part of the gene (del1: bp no. 1000- bp 1080; del2: bp no. 1027- bp1080; and del3: bp 1054-bp 1080). the deletions were made such that there exists no frame shift and the gene translates normally. these 'del 1,2,3' constructs have been tagged with T7 tag at the N-terminus. Also, the T7 tag is in frame with the rest of the gene Also a dry run on 'ORF finder' gives a full length sequence of the translated protein which matches the desired protein sequence (elimination the frame shift problem and n.on-sense mutation incorporation during cloning).
the del 1,2 &3 constructs are in pcDNA3.1 and so I believe they should be well expressed in all cell types.
I have carried out transfection of these constructs but haven't been able to see the expression of T7 tag or the mutated protein on a western blot. the transfection efficiency is about 60-70% (as checked with a GFP plasmid)

Suggestions please...

#2 phage434

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Posted 05 July 2011 - 04:53 PM

1. There is no such thing as "in frame" with respect to the T7 promoter. It drives transcription, not translation, and there is no framing in transcription.

2. What are you transfecting into? People usually speak of transfection when talking about eukaryotic cells. T7 will not transcribe at all in the context of a eukaryotic cell. The T7 promoter is designed to express in bacterial cells containing the gene for T7 RNA polymerase, often in the BL21(DE3) strain of E. coli.

#3 Bomber

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Posted 06 July 2011 - 12:33 AM

if u subcloned it into pcDNA3.1 it should be expressed in any standard cell line given the correct orientation towards the promoter.
another idea would be that the protein is highly unstable due to your modification and therefore you don't see much on the gel.
i experienced that once, though the deletion i had was bigger - but anyhow.
i would suggest to check the expression on RNA level and at the same time check the expression of the protein again. i fyou have strong upregulation on RNA level than i think the idea with protein stability has a bit of support.
in addition it might also depend which protein you are working on and how you extract it from cells.
you could also try to overexpress your protein and check it via confocal or fluorescence microscopy.
did you also tag your wild-type variant of the protein and do you see anything there?
did you try to tag the protein c-terminally?

Edited by Bomber, 06 July 2011 - 12:36 AM.


#4 floxed

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Posted 07 July 2011 - 03:09 PM

1. There is no such thing as "in frame" with respect to the T7 promoter. It drives transcription, not translation, and there is no framing in transcription.

2. What are you transfecting into? People usually speak of transfection when talking about eukaryotic cells. T7 will not transcribe at all in the context of a eukaryotic cell. The T7 promoter is designed to express in bacterial cells containing the gene for T7 RNA polymerase, often in the BL21(DE3) strain of E. coli.



1. its not t7 promoter... its T7 tag... like myc tag...flag tag...
2. the T7 tag usually expresses in eukaryotic cells upon transfection

#5 floxed

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Posted 07 July 2011 - 03:14 PM

if u subcloned it into pcDNA3.1 it should be expressed in any standard cell line given the correct orientation towards the promoter.
another idea would be that the protein is highly unstable due to your modification and therefore you don't see much on the gel.
i experienced that once, though the deletion i had was bigger - but anyhow.
i would suggest to check the expression on RNA level and at the same time check the expression of the protein again. i fyou have strong upregulation on RNA level than i think the idea with protein stability has a bit of support.
in addition it might also depend which protein you are working on and how you extract it from cells.
you could also try to overexpress your protein and check it via confocal or fluorescence microscopy.
did you also tag your wild-type variant of the protein and do you see anything there?
did you try to tag the protein c-terminally?



The stability of the protein was one point where I was stuck too. However, the wild type variant is tagged with T7 at the N terminus too and expresses well. Also if the protein was unstable, it would give atleast faint traces on the western blotting.
I am now working on checking the RNA expression, I hope there is some expression. :D
I haven't checked with tag at the C terminus though. Do you think I should try that since the T7 tag is at the N terminus in the WT and expresses well?

#6 Bomber

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Posted 12 July 2011 - 01:29 AM

if your wil-type variant is n-terminally tagged and expresses well i would not tag c-terminally unless u want to do all variants in a c-tmerinal manner.
regarding the stability of the protein - you might be right with a faint signal on the blot, though i can only tell you that a variant that i once had in my hands was simply not there :-).... so, I guess checking the RNA might indeed be a good control to check things are expressed fine in your system.
on the other hand what u might want to try is to inhibit protein degradation; I could imiagine that due to the aberrant modification of your protein the cells might simply get rid of it by enhanced degradation or something. maybe inhibiting the proteasome by some inhibitors might be another option here...?

good luck

#7 floxed

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Posted 13 July 2011 - 07:35 AM

if your wil-type variant is n-terminally tagged and expresses well i would not tag c-terminally unless u want to do all variants in a c-tmerinal manner.
regarding the stability of the protein - you might be right with a faint signal on the blot, though i can only tell you that a variant that i once had in my hands was simply not there :-).... so, I guess checking the RNA might indeed be a good control to check things are expressed fine in your system.
on the other hand what u might want to try is to inhibit protein degradation; I could imiagine that due to the aberrant modification of your protein the cells might simply get rid of it by enhanced degradation or something. maybe inhibiting the proteasome by some inhibitors might be another option here...?

good luck



Alright, I will try using some protease inhibitors as well. I think there is a ray of hope here. :)

Edited by floxed, 13 July 2011 - 07:35 AM.





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