I have cloned my gene of interest (WT) in a vector and in the same vector with same restriction sites I have cloned the point mutated gene. I am not getting any expression for the WT whereas the mutant protein is expressing fine. DNA quantity and transfection conditions are same. Also in between mutants there is differential expression. Can anyone think of any reason why the expression would be different?
If you made those mutants by site directed mutagenesis from the WT you are using, then no need to sequence the WT. But if you made WT and mutants as separate cloning attempts into the plasmid, then sequence it especially at the gene to plasmid border to see if it is in frame with start codon and stop codon. If you're a beginner in cloning, I would suggest sequence verification.
Next, what type of mutants they are? If Serines/theronines/tyrosines/ are mutated, then it may block the phosphorylation dependent degradation. In that case, your WT might be constantly degraded. To check this, do a MG132 mediated proteasome inhibition of WT transfected cells to see whether MG132 can restore WT expression.
If lysines are mutated, then it might affect ubiquitin dependent degradation of mutant clones. MG132 experiment applies here too.
If it is truncated mutations, then it may have a chance to lose critical residues that determine stability.
Since these WT and mutants are from same type of plasmid, I don't think UTR mediated mRNA stability has to do anything with expression level differences (also excludes RNA interference, translation difference etc). So, the effect has to be post-translational.
If you were looking for expression by Western blotting for the protein, my guess would be that what you are seeing is protein instability related to the induced mutations. As far as the wild type goes, have you sequenced across the entire gene (and flanking plasmid) to confirm that there were no introduced mutations and that the gene has been cloned in the position you had intended? If the wild type is ok, then perhaps try a plasmid loss experiment to see if for some reason the cells are having trouble maintaining it; this can happen especially when expressing replication-associated genes with tags or mutations wherein introduced defects to replication lead to increased plasmid loss.
Dear Rowlands,
I'm checking the expression by Western blotting. But the confusing part is WT is not expressing whereas mutants are expressing fine! So I do not think it is due to the stability of proteins. Even with the mutant proteins, two point mutants have different levels of expression.
As for the plasmid, the vector is commonly used by us, but it has never been an issue. I'll still try to figure out if there is any plasmid loss. But I do not understand why the WT would not express when the mutants are expressing fine.
If you made those mutants by site directed mutagenesis from the WT you are using, then no need to sequence the WT. But if you made WT and mutants as separate cloning attempts into the plasmid, then sequence it especially at the gene to plasmid border to see if it is in frame with start codon and stop codon. If you're a beginner in cloning, I would suggest sequence verification.
Next, what type of mutants they are? If Serines/theronines/tyrosines/ are mutated, then it may block the phosphorylation dependent degradation. In that case, your WT might be constantly degraded. To check this, do a MG132 mediated proteasome inhibition of WT transfected cells to see whether MG132 can restore WT expression.
If lysines are mutated, then it might affect ubiquitin dependent degradation of mutant clones. MG132 experiment applies here too.
If it is truncated mutations, then it may have a chance to lose critical residues that determine stability.
Since these WT and mutants are from same type of plasmid, I don't think UTR mediated mRNA stability has to do anything with expression level differences (also excludes RNA interference, translation difference etc). So, the effect has to be post-translational.
I would sequence the inserts you have cloned into all vectors and make sure you use primers that allow sequencing of the vector-insert boundaries. If you have done site directed mutagenesis in a separate vector then cloned the insert into the vector for expression studies, it might be that the insert has ligated in the reverse orientation (depends on the RE sites you used) or as multiple copies (which would usually be unstable but could interfere with expression).
Are you antibiotic selecting clones after transfecting or are you measuring expression simply on transiently transfected cells? If you are doing the former then clones can have different expression levels depending on the copy number of plasmids taken up by the transfected cell and the site(s) of integration, if it's an integrating vector.
Thanks Jason King!
1. I used different RE for directional cloning. But multiple copies could be a possibility.
2. I'm just checking the transient expression after confirmation of which I'll go for stable transfection.
Thanks for your answer Jinesh,
1. I had the WT as well as the mutant genes cloned in the same vector but with a different C-terminal tag. I used the previous clone for PCR amplification of the same gene but with a new reverse primer now having my desired tag. The previous ones expressed just fine! The tag should not specifically make any difference to the stability of WT protein only. I will consider the phosphorylation dependent degradation hypothesis. Thanks again.
My question is:
Is it a protein from another species that u r expressing?
The expressing organism may see it as foreign. During protein folding due to post translational or co-translational phosphorylation aberrant phosphorylation may happen. In result the migration may be different in native gel or may happen due to molecular wt difference (aberrant phosphorylation) in denaturing gel.
This happened to me as I used to work with a WT and it's mutants althrough out. As someone said earlier post translational and co-translational modifications depend on the secondary structure attained subsequently and simultaneously as well as the sequence. Array of modifications occur depending on it be it a phosphorylation, methylation whatsoever.
My suggestion is if you don't really need to know about it please skip over look this part. LC/MS/MS can give you the perfect answer.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning