How to check the translation? - His-tagged protein expression (Jul/28/2009 )
Hello
It's been a while since I'm trying to resolve the problem, which is quite crucial for my current project. I need to express the bacterial protein in the eukaryotic cells (standart cell culture or in vivo transfection) using tha plasmid vector. I know several publications, in which it is stated that this protein can be expressed ans is even functional in eukaryotic cells. But I can not archieve it with my manipulations!!
What I've done: TA cloned the PCR product, carrying the gene of interest from the storage plasmid into the pcDNA3.1/V5 His TOPO/ vector. I've confirmed, that the insert is correct by restriction analysis and by the sequencing of the insert. Theoretically, the protein should be expressed under the CMV promoter and should have the V5-His tag on the end (I've checked the reading frame for numerous times - it SHOULD be ok..). The RT-PCR from DNAse treated RNA reveales that the gene is transcribed at least. But I do not get any signal with Western Blotting (using anti-His tag antibodies, as there are no antibodies against my protein in the market) and the activity tests (as the protein sholud lead to accumulation of some specific product in the cell) are also negative.
My questions are: is it possible that my gene is trancribed but not translated? if so - how can I verify it?
Or may be there is just something that I've missed?
I will be happy to get any idea how to solve this puzzle!!
ps the gene begins with gccATGGAC, so I did not insert additional Kozak in front of it..
-lna-
lna on Jul 28 2009, 08:05 AM said:
Hello
It's been a while since I'm trying to resolve the problem, which is quite crucial for my current project. I need to express the bacterial protein in the eukaryotic cells (standart cell culture or in vivo transfection) using tha plasmid vector. I know several publications, in which it is stated that this protein can be expressed ans is even functional in eukaryotic cells. But I can not archieve it with my manipulations!!
What I've done: TA cloned the PCR product, carrying the gene of interest from the storage plasmid into the pcDNA3.1/V5 His TOPO/ vector. I've confirmed, that the insert is correct by restriction analysis and by the sequencing of the insert. Theoretically, the protein should be expressed under the CMV promoter and should have the V5-His tag on the end (I've checked the reading frame for numerous times - it SHOULD be ok..). The RT-PCR from DNAse treated RNA reveales that the gene is transcribed at least. But I do not get any signal with Western Blotting (using anti-His tag antibodies, as there are no antibodies against my protein in the market) and the activity tests (as the protein sholud lead to accumulation of some specific product in the cell) are also negative.
My questions are: is it possible that my gene is trancribed but not translated? if so - how can I verify it?
Or may be there is just something that I've missed?
I will be happy to get any idea how to solve this puzzle!!
ps the gene begins with gccATGGAC, so I did not insert additional Kozak in front of it..
It's been a while since I'm trying to resolve the problem, which is quite crucial for my current project. I need to express the bacterial protein in the eukaryotic cells (standart cell culture or in vivo transfection) using tha plasmid vector. I know several publications, in which it is stated that this protein can be expressed ans is even functional in eukaryotic cells. But I can not archieve it with my manipulations!!
What I've done: TA cloned the PCR product, carrying the gene of interest from the storage plasmid into the pcDNA3.1/V5 His TOPO/ vector. I've confirmed, that the insert is correct by restriction analysis and by the sequencing of the insert. Theoretically, the protein should be expressed under the CMV promoter and should have the V5-His tag on the end (I've checked the reading frame for numerous times - it SHOULD be ok..). The RT-PCR from DNAse treated RNA reveales that the gene is transcribed at least. But I do not get any signal with Western Blotting (using anti-His tag antibodies, as there are no antibodies against my protein in the market) and the activity tests (as the protein sholud lead to accumulation of some specific product in the cell) are also negative.
My questions are: is it possible that my gene is trancribed but not translated? if so - how can I verify it?
Or may be there is just something that I've missed?
I will be happy to get any idea how to solve this puzzle!!
ps the gene begins with gccATGGAC, so I did not insert additional Kozak in front of it..
Since this is a bacterial gene, there may be a codon usage problem in a mammalian system that is preventing translation from the transcript. Have you checked your sequence against a mammalian codon usage table? If codon usage is not the problem:
Since you did qRT-PCR, how does your level of transcript compare to other targets? Should there be small amounts of protein? If so, this is probably a sensitivity issue. How are you detecting translation? by Western blotting from whole cell lysates? Are you staining against His or V5? In my experience, V5 is the most sensitive of the two and Westerns should detect even small amounts of protein. How are you visualizing your antibodies? If by ECL, there are more sensitive versions (such as Pierce's femto ECL) made to detect even ng amounts of protein. Is the protein cytoplasmic or nuclear? If nuclear, you can always try nuclear extracts to increase sensitivity by Western blotting. Alternatively, try generating protein by in vitro transciption/translation (such as Promega's TNT kits). I believe that pcDNA3.1 has T7/T3 sites, yes? TNT reactions yield very large amounts of protein and even 2-3 ul of this reaction should lead to intense staining by Western blotting using V5.
-Dr Teeth-
Dr Teeth on Jul 28 2009, 02:55 PM said:
Since this is a bacterial gene, there may be a codon usage problem in a mammalian system that is preventing translation from the transcript. Have you checked your sequence against a mammalian codon usage table? If codon usage is not the problem:
Since you did qRT-PCR, how does your level of transcript compare to other targets? Should there be small amounts of protein? If so, this is probably a sensitivity issue. How are you detecting translation? by Western blotting from whole cell lysates? Are you staining against His or V5? In my experience, V5 is the most sensitive of the two and Westerns should detect even small amounts of protein. How are you visualizing your antibodies? If by ECL, there are more sensitive versions (such as Pierce's femto ECL) made to detect even ng amounts of protein. Is the protein cytoplasmic or nuclear? If nuclear, you can always try nuclear extracts to increase sensitivity by Western blotting. Alternatively, try generating protein by in vitro transciption/translation (such as Promega's TNT kits). I believe that pcDNA3.1 has T7/T3 sites, yes? TNT reactions yield very large amounts of protein and even 2-3 ul of this reaction should lead to intense staining by Western blotting using V5.
Since you did qRT-PCR, how does your level of transcript compare to other targets? Should there be small amounts of protein? If so, this is probably a sensitivity issue. How are you detecting translation? by Western blotting from whole cell lysates? Are you staining against His or V5? In my experience, V5 is the most sensitive of the two and Westerns should detect even small amounts of protein. How are you visualizing your antibodies? If by ECL, there are more sensitive versions (such as Pierce's femto ECL) made to detect even ng amounts of protein. Is the protein cytoplasmic or nuclear? If nuclear, you can always try nuclear extracts to increase sensitivity by Western blotting. Alternatively, try generating protein by in vitro transciption/translation (such as Promega's TNT kits). I believe that pcDNA3.1 has T7/T3 sites, yes? TNT reactions yield very large amounts of protein and even 2-3 ul of this reaction should lead to intense staining by Western blotting using V5.
Thank you very much for the reply! Especially for the advice on Promega kit -I will try to find it, as it really can answer my question (yes, there should be T7 in pcDNA3.1!!)!!
As I have some information on the results already published with the utilization of my gene in eukaryotic cells, I believe that there is no codon usage problem. Anyway, if you know any online source which allows quickly to compare the sequences against the prokaryotic and eukaryotic codons table I would really like to pin it to my favourites (:
Another thing is that as I'm using the strong CMV promoter I expect to get higher expression of the protein of interest. In fact, low expression situation in this case is just the same as no expression situation for me. I agree with you that anti-V5 would be more sensitive, but as I really need the high expression, detecting some traces of the protein might be of no value for my further manipulations. Though it could prove that the translation is working at lest for my transcript.. And for the visualisation I am using the Millipore ECL kit, which is quite sensitive (and the samples for WB are indeed from the total cell lysate).
In fact, what I want to find out is - if I have the problems with translation of my gene, why it could be and how can I get the high expression?
-lna-
lna on Jul 28 2009, 08:05 AM said:
I've confirmed, that the insert is correct by restriction analysis and by the sequencing of the insert.
Did you sequence the insert in both directions? Is the sequence of your gene (from the same genus/species/strain) publicly available? Does your sequence agree exactly with the publicly available one?
lna on Jul 29 2009, 05:04 AM said:
As I have some information on the results already published with the utilization of my gene in eukaryotic cells, I believe that there is no codon usage problem.
Do the published reports use the exact gene you're using (recovered from the same genus/species/strain)? Do the published reports use the same vector? Do they express this gene in the same eukaryotic cells you're using, and under the same conditions?
Can you write to the corresponding author of one of these papers and get a copy of their plasmid to use in your experiments as a positive control?
-HomeBrew-
HomeBrew on Jul 29 2009, 04:10 PM said:
lna on Jul 28 2009, 08:05 AM said:
I've confirmed, that the insert is correct by restriction analysis and by the sequencing of the insert.
Did you sequence the insert in both directions? Is the sequence of your gene (from the same genus/species/strain) publicly available? Does your sequence agree exactly with the publicly available one?
lna on Jul 29 2009, 05:04 AM said:
As I have some information on the results already published with the utilization of my gene in eukaryotic cells, I believe that there is no codon usage problem.
Do the published reports use the exact gene you're using (recovered from the same genus/species/strain)? Do the published reports use the same vector? Do they express this gene in the same eukaryotic cells you're using, and under the same conditions?
Can you write to the corresponding author of one of these papers and get a copy of their plasmid to use in your experiments as a positive control?
in general, the answer is YES (:
in fact, the initial storage plasmid with the gene of interest comes from the same source as the one, used in publication. my sequencing (both directions, certainly) confirmed that my insert is the same as in the sheet, provided with the plasmid. The authors of the publication also used the CMV promoter, but they applied the doxycycline-regulated viral vector for the cells transfection. Currently we prefer to use our transfection methods instead of viral and I can state that it is working quite well with the beta-galactosidase encoding plasmid (the sizes of plasmids are almost the same for my gene of interest..).
-lna-
Can you do pcr on the genomic DNA from the recombinant cells with primers specific for the interior of your gene? You may randomly pick up random light banding, but if you're recombinant your DNA band should be the most intense!
-BIOKMST-
lna on Jul 29 2009, 05:04 AM said:
Dr Teeth on Jul 28 2009, 02:55 PM said:
Since this is a bacterial gene, there may be a codon usage problem in a mammalian system that is preventing translation from the transcript. Have you checked your sequence against a mammalian codon usage table? If codon usage is not the problem:
Since you did qRT-PCR, how does your level of transcript compare to other targets? Should there be small amounts of protein? If so, this is probably a sensitivity issue. How are you detecting translation? by Western blotting from whole cell lysates? Are you staining against His or V5? In my experience, V5 is the most sensitive of the two and Westerns should detect even small amounts of protein. How are you visualizing your antibodies? If by ECL, there are more sensitive versions (such as Pierce's femto ECL) made to detect even ng amounts of protein. Is the protein cytoplasmic or nuclear? If nuclear, you can always try nuclear extracts to increase sensitivity by Western blotting. Alternatively, try generating protein by in vitro transciption/translation (such as Promega's TNT kits). I believe that pcDNA3.1 has T7/T3 sites, yes? TNT reactions yield very large amounts of protein and even 2-3 ul of this reaction should lead to intense staining by Western blotting using V5.
Since you did qRT-PCR, how does your level of transcript compare to other targets? Should there be small amounts of protein? If so, this is probably a sensitivity issue. How are you detecting translation? by Western blotting from whole cell lysates? Are you staining against His or V5? In my experience, V5 is the most sensitive of the two and Westerns should detect even small amounts of protein. How are you visualizing your antibodies? If by ECL, there are more sensitive versions (such as Pierce's femto ECL) made to detect even ng amounts of protein. Is the protein cytoplasmic or nuclear? If nuclear, you can always try nuclear extracts to increase sensitivity by Western blotting. Alternatively, try generating protein by in vitro transciption/translation (such as Promega's TNT kits). I believe that pcDNA3.1 has T7/T3 sites, yes? TNT reactions yield very large amounts of protein and even 2-3 ul of this reaction should lead to intense staining by Western blotting using V5.
Thank you very much for the reply! Especially for the advice on Promega kit -I will try to find it, as it really can answer my question (yes, there should be T7 in pcDNA3.1!!)!!
As I have some information on the results already published with the utilization of my gene in eukaryotic cells, I believe that there is no codon usage problem. Anyway, if you know any online source which allows quickly to compare the sequences against the prokaryotic and eukaryotic codons table I would really like to pin it to my favourites (:
Another thing is that as I'm using the strong CMV promoter I expect to get higher expression of the protein of interest. In fact, low expression situation in this case is just the same as no expression situation for me. I agree with you that anti-V5 would be more sensitive, but as I really need the high expression, detecting some traces of the protein might be of no value for my further manipulations. Though it could prove that the translation is working at lest for my transcript.. And for the visualisation I am using the Millipore ECL kit, which is quite sensitive (and the samples for WB are indeed from the total cell lysate).
In fact, what I want to find out is - if I have the problems with translation of my gene, why it could be and how can I get the high expression?
See http://gcua.schoedl.de/ for various codon usage programs. Click on "each codon vs. usage table" to compare a sequence against various prokaryotic and eukaryotic codon usage databases.
-Dr Teeth-
Dr Teeth on Jul 30 2009, 09:31 PM said:
See http://gcua.schoedl.de/ for various codon usage programs. Click on "each codon vs. usage table" to compare a sequence against various prokaryotic and eukaryotic codon usage databases.
thank you for the link!! if you just could be so kind to give me a hint how to interpretate the results of this analysis (: I'm really puzzled - where to decide that the sequence is definitely not good for mammalian expression? (syrian hamster or human)
-lna-
BIOKMST on Jul 30 2009, 08:19 PM said:
Can you do pcr on the genomic DNA from the recombinant cells with primers specific for the interior of your gene? You may randomly pick up random light banding, but if you're recombinant your DNA band should be the most intense!
I'm so sorry! but I do not get your idea
what are you suggesting actually? -lna-