We expressed a recombinant protein in yeast. This protein naturally has one glycosylation site, but site-directed mutation was done to produce a non-glycosylated form since yeast is able to process this kind of PTM. However, upon analysis of final sample in SDS-PAGE together with the non-glycosylated standard (E. coli-expressed, no yeast-expressed non-glycosylated protein is available for this isoform so far), it revealed that molecular weight of my POI is different (a little bigger) from the standard. I did HPLC analysis of my POI and compared with the standard and again they have different retention time (earlier than the standard). Further, my POI has two peaks in it. Bioactivity was also checked, but the activity of my POI is quite comparable to the standard.
We were contemplating of the fact that it was produced in a different expression host, so maybe something might have influenced it, but as to what it is, I am really still lacking in knowledge.
Any ideas and suggestions would greatly help.
Try mass spectrometry if you know more and having a good resolution and precision on the mass. (LC-ESI-TOF for example). Very fast and require low volume
It could be possible that you have a mixture of glycosylated and "nonglycosylated" protein in your sample. By convention, the glycosylated protein may be more polar than the nonglyco.. one and may elute out first if doing reverse-phase hplc and also giving you two peaks.. This mixture on SDS-PAGE may appear as one band.. The size difference from the standarad on SDS-PAGE alone could be due to so many reasons.. best to compare on mass spec if you need to confirm all of this..
If you suspect that there may be a difference in the sequence or anything.. you can probably try to isolate your sequence from total RNA content and compare it with the standard and others
Hope this helps.. Good luck
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