I'm working on a certain protein (protein A). It has been constructed on a plasmid, and two point mutations have been created (WT and mutant 1 and 2). After transfecting cells with equal amounts of plasmid DNA, I found that gene expression of protein A in WT is twice that of mutant 2 and slightly higher than that of mutant 1. I've repeated the same experiment 4 times and got the same result. I tried another batch of plasmid newly extracted fro E. Coli., same result. Is this a common phenomenon? What would be causing this? Is it meaningful to find out why and if so, how can I step by step figure it out?
Hello Paikwin Li,
A likely explanation is that the mutations affect the stability of the protein and make it more susceptible to destruction by proteases; it could also be that the mutations afffect in some way the structure of the mRNA, which may influence the efficiency of translation
Dear Paikwin Li
Whether the increase in protein A expression was observed as a protein level or as an mRNA level, the assumed reason would be different.
From your text, I read that the mRNA amount increased. My guess is that there is a target sequence for the miRNA, and the mutation made it easier to be degraded. Couldn't this be inferred by changing to a different sequence from the current one or by predicting the miRNA sequence targeting Protein A?
If you have observed an increase in the amount of protein, then as Dr. Pierre Beguin says, Protein A is more susceptible to proteolysis due to the mutation. For example, it could be a mutation that makes it ubiquitinated.
Best,
Missense mutations should not differ the gene expression from plasmids. If the results are consistent, then I think you should dilute the samples differently to get similar band intensity in your blots, use those dilutions for your downstream experiments.
When you say gene expression, do you mean mRNA or protein? Also, what sort of cells are you transfecting, eukaryotic or prokaryotic? The gene regulation mechanisms are different enough that it would be helpful to know.
Do the mutations alter the peptide length or are they an amino acid alteration? If it is a changed amino acid, it is to a more rare codon?
Dear Amy Klocko , thank you for your reply. I meant mRNA level. The expression difference was observed in Q-PCR. I used cancer cell lines to do transfection. The mutations are missense mutations, so both mutants have one animo acid changed into another one. As for rare codons, I think it's a good point to consider; however, as only one amino acid is altered, to my understanding, the impact on gene expression should be limited? Please share more of your thoughts.
Dear Papri Basak , yes, I have done WB post transfection, though the expression result here refered to outcome of Q-PCR. As for WB, I haven't yet noticed much difference between WT and the mutants. But I'll repeat the experiment to see whether there is change between WT and mutants on the protein level.
Dear Pierre Béguin , the difference mentioned is from mRNA in Q-PCR. But in WB, no significant change is yet observed. Regarding your hypothesis that mutations may render the protein more susceptible to destruction by proteases, could it be the contrary, that the mutated protein is more stable and through some sort of feedback regulation, less mRNA of the mutated gene is produced?
Dear Narumi Uno , thank you for your detailed reply. Yes, it is mRNA that has increased in WT compared to the mutants. I agree that it may have something to do with miRNA and that the mutation may render it easier for degradation. By saying "Couldn't this be inferred by changing to a different sequence from the current one", do you mean changing the position of the primer set on mRNA for Q-PCR?
This is a vague image, but if I assume that miRNAs are the culprit, since the target is only about 20 nucleotides, I may prepare a large number of mutant forms by referring to the current Protein A mutants 1 and 2 and replacing all of the surrounding 20 nts as much as possible with silent mutations to identify the miRNA target sequence.
I suppose.......
You can discuss this with Dr. Google.
It is pretty surprising to find a single nt changing affecting RNA levels. It is highly unlikely that this is due to some feedback from the mutant protein. You could probably figure out the cause of the difference, but is it really worth the time you would spend?
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