Dear all, is there any possibility that C-myc epitope tags at recombinant protein can cause the protein degradation/failure in protein expression ? I can't find any previous literature that can support the failure of protein expression due to c-myc.
The lab work has already stopped after a countless of optimization been performed to express the protein.
Regards,
What organism are you expressing the protein in, and do you verify expression by western blots? If so, antibodies can be very crucial. You could try looking for the said protein by using an antibody that recognises the protein itself, rather than myc.
Hi Dr Rashmi Ramesh , my protein was expressed using eukaryotic yeast Pichia pastoris. For your information, the proteins size that I tried to express are 42 kda, 31 kda, 26 kda, 19 kda and 15 kda (they were derived from the same structure ectodomain). I successfully expressed the 42kda protein. Unexpectedly, the other 4 proteins were failed to expressed using the same protocol/procedure. All these proteins were 6xHis-tagged recombinant proteins. 42 kda proteins were successfully expressed and purified for downstream assay. 42kda protein was confirmed through anti-His western blot.
I also did analyze 5 recombinants for the correctly sized PCR product to rule out the possibility that colonies that i picked to express was problematic.
In yeast, you have to account for proteasomal (ubiquitin dependent) degradation of unstable, misfolded or aggregated proteins. Floppy tags may also affect the efficiency with which a given protein is degraded.
Hi Aina,
According to my experience, truncated fragments may be expressed but degraded due to unstable structure during the experiment. It is recommended to try different tags, truncation, or expression systems. Using proper tags can prevent protein degradation and help dissolution. I used to express a protein that is easily degraded and found that the probability of protein degradation in the N-His construct is relatively high, and the two-terminal-His tags can effectively prevent protein degradation.
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