I have cloned my genes in a CMV vector and transfected them in HEK293 cells. After harvesting the cells, the expression level at the transcriptional level is increased compared to the control well. However, the peptide sequence is not seen to be expressed as analyzed by mass spectrometry.
Can you please give an insight into the possible reason for the same?
Thank you.
Hello,
if you can detect mRNA expression but no protein production at all there is a possibility that you damaged the vector backbone while cloning so the transcript lacks the necessary instructions for nuclear export and translation usually found on 3`end of MCS. Try sequencing the plasmid backbone and comparing it to the original sequence.
Mateo Glumac Thank you for your insight. I have done the sequencing, and the results are identical to the original sequence.
It sounds like you're observing an increase in transcriptional expression without a corresponding increase in the expression of the peptide sequence as analyzed by mass spectrometry. There are several potential reasons for this discrepancy:
Post-Transcriptional Regulation: The increased transcriptional expression may be subject to post-transcriptional regulation, such as mRNA degradation, alternative splicing, or microRNA-mediated regulation, which could prevent the translation of the mRNA into the peptide sequence.
Protein Degradation: The expressed peptide sequence may be subject to rapid degradation, leading to undetectable levels by mass spectrometry. This could be due to instability of the protein or increased proteasomal degradation.
Mislocalization or Secretion: The expressed peptide sequence might not be retained within the cell and may be secreted, mislocalized, or targeted for degradation before mass spectrometry analysis.
Translational Regulation: The translation of the mRNA into the peptide sequence may be regulated by factors such as RNA-binding proteins, translational repressors, or other post-transcriptional modifications.
Experimental Artifacts: It's also important to consider potential experimental artifacts, such as issues with sample preparation, mass spectrometry analysis, or antibody specificity.
To address this issue, it may be helpful to perform additional experiments to investigate the translational regulation and stability of the expressed peptide sequence. This could include examining the mRNA stability, protein localization, and investigating potential post-translational modifications that may affect protein stability and detection.
Hi again,
did you try WB or did you only use MS?
If you can't detect protein production by WB you should clone your peptide into a different plasmid vector.
Mateo Glumac Unfortunately, Western blotting could not be opted as commercial antibodies are not available for the desired peptide sequence.
Hi,
how long is the sequence? Is it completely synthetic or is it a part of an already known protein? What do you want to achieve with your experiment, should your peptide act as a ligand? Is tagging it with His, FLAG, or MYC tag an option? Insertion of these tags can be done by a simple PCR.
Mateo Glumac
The peptide sequence is 101 a.a long. It is already a domain of known protein. We want to use our peptide as an antigen. We have tagging the protein with His tag in the pipeline, but was keen to know whether the protein is being synthesized or not first.
Hi,
if you are using it as an antigen it should be cell membrane-bound. Membrane-bound proteins are notoriously hard to solubilize for analysis. This paper dealt with this problem:
Article Improved method for the analysis of membrane proteins by mas...
Hi,
some expression vectors also carry sequences for in vitro cell-free translations. If your vector does not have it, try different one with these additional pieces. It will allow you to have your peptide or, at least, will show that problem with expression (on any level) in original setting. Good luck.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line