10 replies to this topic

[Drake1982]

Hi,
I have a question regarding a YFP-protein fusion expression:
I cloned YFP under the p(BAD) promoter to have a controlled expression of YFP in Ecoli. Usually, I induce the plasmid with 0.1% L-ara at an OD of 0.4. This actually works nicely.
In the next step, I cloned my protein of interest to the N-terminal domain of YFP; and everything again under the p(BAD) promoter. Induction of this construct led to a spot accumulation in the cells.
Actually, in most cases it only forms one spot per cell.

There are many different statements online discussing such weird behavior. One e.g. says that GFP does not fluorescent in inclusion bodies anymore. How trustful is this statement? Might this also be true for YFP?

Furthermore, I tried to visualize an over-expression of YFP as well as the YFP-protein fusion on a SDS gel. I was able to identify an over-expressed band at 27kDa for YFP; but unfortunately, I could not get an over-expressed band for the fusion. Is this another indication for inclusion bodies?

Is it possible that my protein is misfolded? I mean, the protein fusion gives me at least some localized fluorescent, so I would say the protein (which is in front of YFP) might also be correctly folded?

Is there any way to rule out the theory of inclusion bodies? What might be an alternative explanation? Could it be that the expressed protein binds somewhere in the cell? Although I cannot say that all spots occur at the same location. Some are at the pole, some are in the middle ...

Any suggestions are welcome!
Thanks!

[proteaMatt]

Drake1982, on 21 April 2011 - 09:55 AM, said:

Furthermore, I tried to visualize an over-expression of YFP as well as the YFP-protein fusion on a SDS gel. I was able to identify an over-expressed band at 27kDa for YFP; but unfortunately, I could not get an over-expressed band for the fusion. Is this another indication for inclusion bodies?

I am curious to know if you can verify (according to the molecular weight marker on the gel) that the protein+YFP should have been able to enter into the gel.

[Drake1982]

proteaMatt, on 25 April 2011 - 05:54 AM, said:

Drake1982, on 21 April 2011 - 09:55 AM, said:

Furthermore, I tried to visualize an over-expression of YFP as well as the YFP-protein fusion on a SDS gel. I was able to identify an over-expressed band at 27kDa for YFP; but unfortunately, I could not get an over-expressed band for the fusion. Is this another indication for inclusion bodies?

I am curious to know if you can verify (according to the molecular weight marker on the gel) that the protein+YFP should have been able to enter into the gel.

I'm not quite sure whether I understand your response.
The thing is, I know the molecular weight of my protein and I know the molecular weight of YFP - overall approximately 60kDa .. and even if it would be dimeric .. I should be able to visualize it on a gel .. no?
Furthermore, what I forgot to mention: I also overexpressed my protein without YFP (at least lets say I tried to ) and run it on a gel. But also in this case, no significant band between induced and uninduced sample was observable. Which again leads me to the question (hypothesis) about inclusion bodies!

[proteaMatt]

Drake1982, on 25 April 2011 - 06:23 AM, said:

proteaMatt, on 25 April 2011 - 05:54 AM, said:

Drake1982, on 21 April 2011 - 09:55 AM, said:

Furthermore, I tried to visualize an over-expression of YFP as well as the YFP-protein fusion on a SDS gel. I was able to identify an over-expressed band at 27kDa for YFP; but unfortunately, I could not get an over-expressed band for the fusion. Is this another indication for inclusion bodies?

I am curious to know if you can verify (according to the molecular weight marker on the gel) that the protein+YFP should have been able to enter into the gel.

I'm not quite sure whether I understand your response.
The thing is, I know the molecular weight of my protein and I know the molecular weight of YFP - overall approximately 60kDa .. and even if it would be dimeric .. I should be able to visualize it on a gel .. no?
Furthermore, what I forgot to mention: I also overexpressed my protein without YFP (at least lets say I tried to ) and run it on a gel. But also in this case, no significant band between induced and uninduced sample was observable. Which again leads me to the question (hypothesis) about inclusion bodies!

I guess my point is:
Reasons why the protein didn't show up on the gel - 1. It wasn't there to begin with; 2. It didn't enter into the gel even though it was present.

Just trying to eliminate reason 2 as a possiblity. My concern was whether or not your protein is too large with the YFP added to it for it to not enter the gel. I'm not sure about the conditions of your gel, but 60 kDa isn't especially large.

Honestly, I can't really help you too much with your overall experiment, except for the phase that I commented on. Just wanted to make sure it wasn't something simple that might be overlooked.

[Drake1982]

proteaMatt, on 25 April 2011 - 09:45 AM, said:

Drake1982, on 25 April 2011 - 06:23 AM, said:

proteaMatt, on 25 April 2011 - 05:54 AM, said:

Drake1982, on 21 April 2011 - 09:55 AM, said:

Furthermore, I tried to visualize an over-expression of YFP as well as the YFP-protein fusion on a SDS gel. I was able to identify an over-expressed band at 27kDa for YFP; but unfortunately, I could not get an over-expressed band for the fusion. Is this another indication for inclusion bodies?

I am curious to know if you can verify (according to the molecular weight marker on the gel) that the protein+YFP should have been able to enter into the gel.

I'm not quite sure whether I understand your response.
The thing is, I know the molecular weight of my protein and I know the molecular weight of YFP - overall approximately 60kDa .. and even if it would be dimeric .. I should be able to visualize it on a gel .. no?
Furthermore, what I forgot to mention: I also overexpressed my protein without YFP (at least lets say I tried to ) and run it on a gel. But also in this case, no significant band between induced and uninduced sample was observable. Which again leads me to the question (hypothesis) about inclusion bodies!

I guess my point is:
Reasons why the protein didn't show up on the gel - 1. It wasn't there to begin with; 2. It didn't enter into the gel even though it was present.

Just trying to eliminate reason 2 as a possiblity. My concern was whether or not your protein is too large with the YFP added to it for it to not enter the gel. I'm not sure about the conditions of your gel, but 60 kDa isn't especially large.

Honestly, I can't really help you too much with your overall experiment, except for the phase that I commented on. Just wanted to make sure it wasn't something simple that might be overlooked.

Any comments are appreciated! Thanks for your input!

[Chris22]

Dear Drake1982

Sorry for replying to an old topic but I am having the exact same problem with a GFP fusion as you described! And although nobody replied to this topic here, I was hoping to get your into as to whether you solved your SDS issue?

My own GFP fusion protein does not show up on any sds gel or western blot using anti-GFP mAb. However, the GFP protein by itself shows up everytime, in both sds and western.

I hope you can help.

Kind Regards
Chris

[Chris22]

Ａｐｏｌｏｇｉｅｓ， Ｉ ｆｏｒｇｏｔ ｔｏ ｍｅｎｔｉｏｎ ｔｈａｔ Ｉ ｉｎｃｌｕｄｅｄ ａ ｌｉｎｋｅｒ， ｃｏｐｉｅｄ ｆｒｏｍ ａ ｃｏｍｍｅｒｉａｌ ＧＦＰ ｖｅｃｔｏｒ， ａｎｄ ｉｔ ｉｓ ａｎ Ｎ－ｔｅｒｍｉｎｉ ＧＦＰ ｆｕｓｉｏｎ。 Ｉ ｈａｖｅ ｓｅｑｕｅｎｃｅｄ ｔｈｅ ｃｏｎｓｔｒｕｃｔ ｔｗｉｃｅ ｔｏ ｃｏｎｆｉｒｍ ｎｏ ｆｒａｍｅ ｓｈｉｆｔ ｏｒ ｄｅｌｅｔｉｏｎ ｈａｓ ｏｃｃｕｒｒｅｄ。

Ｉ ａｌｍｏｓｔ ｅｘｐｅｃｔ ｉｔ ｔｏ ｆｏｒｍ ｉｎｃｌｕｓｉｏｎ ｂｏｄｉｅｓ ｄｕｅ ｔｏ ｔｈｅ ｎａｔｕｒｅ ｏｆ ｔｈｅ ｐｒｏｔｅｉｎ， ｈｏｗｅｖｅｒ， Ｉｓ ｉｔ ｐｏｓｓｉｂｌｅ ｔｏ ｓｔｉｌｌ ｓｅｐａｒａｔｅ ａｎｄ ｖｉｓｕａｌｉｓｅ ａ ｆｕｓｉｏｎ ｕｓｉｎｇ ＳＤＳ ａｎｄ ｗｅｓｔｅｒｎ ｗｈｅｎ ｉｔ ｉｓ ｆｏｒｍｉｎｇ ｉｎｃｌｕｓｉｏｎ ｂｏｄｉｅｓ？

Ｉ ｃｏｕｌｄ ｓｗｉｔｃｈ ｔｏ  Ｈｉｓ－ｔａｇ ｂｕｔ ｔｈｅ ｉｓｓｕｅ ｍａｙ ｓｔｉｌｌ ｂｅ ｔｈｅ ｓａｍｅ ａｎｄ ｔｉｍｅ ｉｓ ｐａｉｎｆｕｌｌｙ ｓｈｏｒｔ。

Ａｎｙ ａｄｖｉｃｅ ｗｏｕｌｄ ｂｅ ｓｏ ｗｅｌｃｏｍｅ！！！

Regards
Chris

[Curtis]

Do you have any antibody for your protein of interest that you can run western blot with that instead of anti-GFP? maybe you need to try that...However, most anti-GFPs are designed quite well and can label GFP even not in original shape.

The other question is that how far is the start codon from your promoter? Don't expect to see a band as intense as the empty vector's band on western blot membrane. The intensity of fused proteins are usually much less than the un-fused GFP! Do you run controls?

[Chris22]

Curtis, on 11 July 2012 - 07:11 PM, said:

Do you have any antibody for your protein of interest that you can run western blot with that instead of anti-GFP? maybe you need to try that...However, most anti-GFPs are designed quite well and can label GFP even not in original shape.

Hi Curtis

Thanks for replying,

I did not have another antibody to use at the time but I am currently cloning another similiar protein to which I have a primary antibody against. This new protein fused to GFP in the same way as the original should tell me if the system as a whole is working or whether it is an issue with the GFP fusion process itself (using both anti-protein and anti-GFP westerns) So essentially doing as you said.

Curtis, on 11 July 2012 - 07:11 PM, said:

The other question is that how far is the start codon from your promoter? Don't expect to see a band as intense as the empty vector's band on western blot membrane. The intensity of fused proteins are usually much less than the un-fused GFP! Do you run controls?

The start codon is SOE'd directly to the first ATG start codon in the vector, approx 5-7bp apart.  It is good to know that I should expect a weaker band, I was expecting this due to the increased size of the protein but will keep it in mind for future westerns, I presume this is also the case for fluorescence plate readers? Maybe this is why I saw no peak for fusions while a sharp peak was seen for GFP alone. Maybe it is a limit of detection issue?

Controls used for all protocols include the Empty vector strain, GFP+ strain alone and the Wildtype strain lacking any modification for good measure.

Kind Regards
Chris

[Curtis]

yes, it must be why the machine reads it lower. Why do you say strain?

[Chris22]

I use "strain" as it is the GFP gene expressed in this particular gram positive strain. The GFP+ strain (just overexpressing GFP). The GFP-fusion+ strain ( protein with GFP fusion) and the wildtype strain (with no vectors associated with overexpression for background).

I include each of these strains in each 96 well plate during readings.
Apologies for the confusion, or if I am using the term incorrectly.

Regards
Chris