Protein Expression

Original Message
From:manju
Date:05/14/2012 15:29:00
Sorry about the confusion. The overexpressed protein is made by truncating 90-95 amino acids from the one end. So theoritically it should run approximately 10 Kd lower than the in-vivo protein on a SDS-PAGE gel. However, when I run the cell lysate (considering there is an in-vivo target protein), and using the overexpressed pure protein as a control, I see that both the lanes have the bands running at the same size.
So, the problem is if I am looking at the right protein or not.
Hope this is more clear.

Much clearer, thanks. Here are a couple thoughts:
How sure are you that the band you see on a western is the protein you think it is? Have you tried pre-incubating the Ab with the antigen protein (or antigen-peptide) before using it to probe the blot? If the Ab is against the target protein of interest, the pre-incubation with the antigen should block, or markedly reduce the signal compared to pre-incubation without the antigen, or with an irrelevant protein.

How big are your proteins? If they are very large (>100 kDa), it can be hard to see a difference of 10 kDa. The ability to see differences in MW will also depend on the type of gel you are running. Gradient gels are particularly good at giving good separation.

Have you verified the construct by sequencing, just to make sure the insert is exactly what you think it is?

Have you tried running a 2D western on the samples? It's possible that the migration of one or both of the proteins doesn't exactly reflect the MW. If this is the case, a 2D gel can usually resolve the proteins into different spots.

Is it possible that the native protein in the lysate is degraded?

Sorry about the confusion. The overexpressed protein is made by truncating 90-95 amino acids from the one end. So theoritically it should run approximately 10 Kd lower than the in-vivo protein on a SDS-PAGE gel. However, when I run the cell lysate (considering there is an in-vivo target protein), and using the overexpressed pure protein as a control, I see that both the lanes have the bands running at the same size.
So, the problem is if I am looking at the right protein or not.
Hope this is more clear.

Original Message
Date:05/05/2012 16:27:00
Hey all,

I have a trouble related to western. I had expressed a pure protein in E.Coli, and we generated an antibody against it. I have the antibody, but now when I run the cell lysate, and use this overexpressed protein as control, they run the same. The overexpressed protein is 10Kd smaller than the in vivo expressed protein. I am not sure if I am looking at the right band. Since the antibody was polyclonal, I had several bands showing up in western, both in the wild type and the insertion-mutant. After purification of the antibody, there is one single band in wild type, and the overexpressed protein and in vivo protein from the wild type cell lysate run at the same position. Please suggest me what can be done.

Having a little trouble understanding the problem.
when I run the cell lysate, and use this overexpressed protein as control, they run the same. The overexpressed protein is 10Kd smaller than the in vivo expressed protein. These statements seem contradictory. If they run the same, how can one be 10 kDa smaller? Does the cell lysate contain the in vivo expressed protein?

the insertion-mutantHow does the insertion mutant fit into this problem. This is the first time it's mentioned and you give no explanation as to what it is, what the insert is, how big the insert is etc.

Basically, I don't know what you are asking. Could you please clarify the problem?

thanks.

Hey all,

I have a trouble related to western. I had expressed a pure protein in E.Coli, and we generated an antibody against it. I have the antibody, but now when I run the cell lysate, and use this overexpressed protein as control, they run the same. The overexpressed protein is 10Kd smaller than the in vivo expressed protein. I am not sure if I am looking at the right band. Since the antibody was polyclonal, I had several bands showing up in western, both in the wild type and the insertion-mutant. After purification of the antibody, there is one single band in wild type, and the overexpressed protein and in vivo protein from the wild type cell lysate run at the same position. Please suggest me what can be done.

Some thoughts: make sure you are using sufficient reducing agent. I usually use a final beta-ME concentration of around 0.36 M. Make the sample buffer fresh each time (i.e. don't add the beta-ME until you are ready to use it). If you have been heating at 95°C, try 60°C, or 37°Ç. If you've already been heating at a lower temp, up it to 95°C. Try running some purified IgG with and without reducing agents to make sure everything is working properly. If none of this works, you might have to try mutating the cysteines to eliminate the chance of disulfides and then see what happens on the gel.

I agree with everything here. Hope it helped you.









Regards,
Laine
[URL=http://prettravaux.net]prêts travaux[/URL]

Hi!
I remember that; the commercial house mentions that this stock can express protein even though not has added inductive

Hey guys. may anybody explain me how I can convert dimer formed by disulfide band to monomer ? I exactly need monomer but after expression I detect on my SDS-PAGE dimer instead of monomer. I have added reducing agents to sampling buffer but it seems that It does not work.
One more question, why the position of bands differ between pre stained marker and common markers(i.e. the position of 17kDband is not the same as 17KD in prestained marker.)
Thank you all

Are you certain that the dimerization is a result of disulfides? If it is, reducing SDS-PAGE should show only monomer. A good example is IgG. Without reducing agents, you see only a band at 150-160 kDa. With reducing agents you see heavy and light chains at 55 and 25 kDa, respectively. In rare instances, some protein-protein interactions remain after heating in sample buffer containing DTT or beta-ME. In these cases, something other than disulfides is usually the case.

Some thoughts: make sure you are using sufficient reducing agent. I usually use a final beta-ME concentration of around 0.36 M. Make the sample buffer fresh each time (i.e. don't add the beta-ME until you are ready to use it). If you have been heating at 95°C, try 60°C, or 37°Ç. If you've already been heating at a lower temp, up it to 95°C. Try running some purified IgG with and without reducing agents to make sure everything is working properly. If none of this works, you might have to try mutating the cysteines to eliminate the chance of disulfides and then see what happens on the gel.

Hey guys. may anybody explain me how I can convert dimer formed by disulfide band to monomer ? I exactly need monomer but after expression I detect on my SDS-PAGE dimer instead of monomer. I have added reducing agents to sampling buffer but it seems that It does not work.
One more question, why the position of bands differ between pre stained marker and common markers(i.e. the position of 17kDband is not the same as 17KD in prestained marker.)
Thank you all

Thank you BlueDevil for your generous help. I will use your comments for continuing my work.

Because my protein has His tag at its N-terminal I have done purification by Ni-NTA spin column. I have got 3 bands! and It seems that I should change the buffer composition.(adding imidazole,..)
Does the presence of a band after purification with spin column mean that it is my protein? Because now I have no anti His tag Ab so I cannot do western and also my protein doesnot have activity.

At some point you're going to have to verify that it is your protein. An antibody against the protein is the most direct way. It's not uncommon for some proteins to bind to a Ni-NTA column if they have a run of histidines. You can't rely solely on the fact that a protein was purified via binding to a Ni-NTA column as proof that the protein is what you think it is. Since you are going to the trouble of expressing and purifying a recombinant protein, I assume you are going to use the protein in experiments. In order to explain any results you may generate, you're going to have to be sure that you are working with the protein you think you are. If an antibody doesn't exist for the protein, your next best bet is to have the purified protein analyzed by a proteomics/mass spectrometry facility.

Because my protein has His tag at its N-terminal I have done purification by Ni-NTA spin column. I have got 3 bands! and It seems that I should change the buffer composition.(adding imidazole,..)
Does the presence of a band after purification with spin column mean that it is my protein? Because now I have no anti His tag Ab so I cannot do western and also my protein doesnot have activity.

Bluedevil thank you for your useful reply. Among induced and uninduced bands there is a band in right size, but the difference of induced and uninduced is the intensity of bands, ie. in uninduced, the band is weak but in induced the band is obvious and high.I was not able to find an obvious difference between empty vector and recombinant!
So If I look only to induced and uninduced bands it seems that i got an expression. But if i include empty vector , the judgment is hard for me. And I don't know what should i do now. Omiting empty vector from my trials or continuging the expression optimization.

I think at this point you need to verify that the band you see is the protein you are hoping to express. This could be either by antibody or an activity assay, if you have one. Otherwise, you might spend valuable time optimizing the expression of an irrelevant protein.

Also, the fact that you see a band at the same MW in the induced and uninduced is nothing to worry about. It could either be a different protein that just happens to run at essentially the same MW as the recombinant or it could be due to a "leaky" inducible system (i.e. you get some expression even without induction).

Bluedevil thank you for your useful reply. Among induced and uninduced bands there is a band in right size, but the difference of induced and uninduced is the intensity of bands, ie. in uninduced, the band is weak but in induced the band is obvious and high.I was not able to find an obvious difference between empty vector and recombinant!
So If I look only to induced and uninduced bands it seems that i got an expression. But if i include empty vector , the judgment is hard for me. And I don't know what should i do now. Omiting empty vector from my trials or continuging the expression optimization.

Hi,
I cloned my insert into pET28 after a His tag codon and decide to express it in E.coli BL21.In order to check its expression,is it necessary to include the cells carrying empty pET28 along with recombinant cells ?I mean should I grow and induce cells having pET28 and check their protein expression pattern in comparison with recombinant s? In SDS-PAGE should I see an obvious difference between proteins of recombinant and others?
I will be grateful if you help me in this case.

You could do it that way (i.e. with the empty vector) but it's not really necessary. I usually just compare induced and uninduced cultures, both carrying the complete construct. Depending on the level of expression, you should see a strong band in the induced cultures compared to the uninduced. But, if you've only just started working with this construct, you may have some optimization to carry out. Things like incubation temperature, concentration of IPTG, length of induction, and strain of bacteria can be very important. Also, if you don't see the band among the soluble proteins, make sure to run some of the pellet also.

Hi,
I cloned my insert into pET28 after a His tag codon and decide to express it in E.coli BL21.In order to check its expression,is it necessary to include the cells carrying empty pET28 along with recombinant cells ?I mean should I grow and induce cells having pET28 and check their protein expression pattern in comparison with recombinant s? In SDS-PAGE should I see an obvious difference between proteins of recombinant and others?
I will be grateful if you help me in this case.

Check out the following thread if you haven't already done so. This topic has come up a couple of times. It can be difficult to express membrane proteins in e.coli (or other bacteria). For some reason, they are either poorly expressed and/or are toxic to the bacteria. In addition, the signal sequence may not be recognized, which means the protein is not corrected targeted to the membrane. I would assume once this happens the protein probably mis-folds and is either degraded or ends up in inclusion bodies. If you are just interested in the cytoplasmic portion of the protein, lets say you wanted to do pull-down assays to look at interaction partners, it would probably be easy to just express this portion of the protein.

As I mentioned, check out this thread and the following links:

http://www.biocompare.com/forums/ViewThread.aspx?threadid=881

http://www.traffic.dk/toolbox/pdfs/2_2.pdf

http://proteinscience.org/cgi/content/full/14/1/148

I think not all Signal peptide can work in e.coli, and not all protein can secrit with same sig peptide. In fact, most of them don't work. I don't know the reason but i am sure its not caused by the his tag. Why not try other expression vector?

Sinobio Biotech Ltd.
Manufacturer of Cytokines

Original Message
From: msaleem
Date: Sunday, May 21, 2006 6:00:39 AM
I am trying to express periplasmic protein with N-terminal seginal sequence from Pesudomonas in E. coli. I tried pET vector with C-terminal his tag. My protein is expressing but I am unable to purify it from periplasmic fraction. Is this problem because of signal sequence from other species or His tag is causing in export of protein to the periplasm? if yes which vector in future should I use?

Regards
Saleem

How do you know your protein is being expressed, is it soluble in the cytoplasm or found in inclusion bodies?

Has a signal sequence from pseudomonas been shown to work in e.coli?

Did you use a bacterial expression vector designed for secreted proteins or just the signal sequence from the gene cloned into a normal his-tag vector?

check out some of these vectors designed for periplasmic expression of proteins in bacteria.

http://www.emdbiosciences.com/html/NVG/solubility.html

http://www.sigmaaldrich.com/img/assets/15040/vectors_new.pdf

Original Message
From: msaleem
Date: Sunday, May 21, 2006 6:00:39 AM
I am trying to express periplasmic protein with N-terminal seginal sequence from Pesudomonas in E. coli. I tried pET vector with C-terminal his tag. My protein is expressing but I am unable to purify it from periplasmic fraction. Is this problem because of signal sequence from other species or His tag is causing in export of protein to the periplasm? if yes which vector in future should I use?

Regards
Saleem