Huge mRNA overexpression, but no protein overexpression. - (Sep/11/2008 )

Hello all,

I transfected my cloned plasmid into HEK 293 cells. There was huge mRNA expression by real time PCR, but there was no protein overexpression. Could you give me any suggestions? Thanks a lot in advance!

dc11

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May be this protein's expression is regulated very strictly or may be is degraded very quickly.

When did you look for protein expression and how did you check expression?

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Hi Scolix,

Thank you for your response. I checked the expression in 48 and 72 hrs using western blotting. The HEK293 cells express the endogenous protein which can also be detected by the antibody (the exogenous protein by the plasmid is rat protein). Do you think it is a problem? Thanks.

dc11

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Hi dc11

I don't think its a problem to express rat protein in HEK 293 cells. Does your protein have an epitope tag? Using an antibody against the epitope tag will help you to differentiate between endogenous and the rat protein.

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Hi Scolix,

My protein does not have an epitope tag. If the cDNA has one or two base changes ( I am not quite sure about that), does it really matter to the expression? Thanks.

dc11

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Hi dc11

It should not matter if there is base change or not unless it changes sequence of aminoacid to stop codon or something else leading to degradation.

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Didnt you confirm the cDNA sequence after you got a high transcription of mRNA but not expression of protein? Think about an ORF shift, although it rarely happens.

Or simply move on and make a new construct, add a flag or myc tag on the protein, that'll be a lot easier.

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If the cDNA has one or two base changes ( I am not quite sure about that), does it really matter to the expression? Thanks.

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Few things. One, you must check the sequence of your cDNA to make sure there isn't a stop codon preventing the production of detectable protein or a frame-shift which is producing a nonsense peptide.

Two, are you sure you have an appropriate Kozac sequence in order to help translation? Generally a Kozac just helps the efficiency but I have seen instances that without it there was no expression.

Last, it may be a problem that you are expressing the rat protein in human cells. Does your antibody specifically state it recognizes rat? Clearly it identifies the human protein since you can see endogenous but it may not recognize your expressed rat. Have you aligned the human and rat sequences to see the conservation within the immunogenic region? If your antibody is rabbit you have a better chance of cross-species reaction but if it's a mouse, the epitope must be conserved.

I would recommend you add an epitope such as HA or myc. This can be done quite easily (one round of pcr with properly designed primers) and then you can use one of the many commercially available antibodies to the tag rather than the protein itself. Otherwise, if available, try your membrane with a different antibody to the specific protein.

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Hi rkay447,

1. I checked the cDNA sequence and it is correct.

2. I have a Kozac sequence in the cDNA.

3. You are very right, I am expressing a rat protein in HEK293. The antibody (rabbit) does detect both rat and human protein. HEK293 itself also expresses the protein. But I think if there is strong overexpression I should be able to see the differences between control vector-transfected and plasmid-transfected HEK293.

4. I am cloning the cDNA in frame with V5/His (which is in the vector) now. I just failed to get the PCR working.

Thank you very much for your great suggestions!

dc11

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Hi Twisters,

I did confirm the cDNA sequence. I am trying to make a new construct which has V5/His tag. Thank you very much!

dc11

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