Since the band below the 55kDa ladder band isn't labelled, I am not able to figure out the scale and don't know which of these bands is your target protein at 44kDa. PTMs like Phosphorylations, glycosylation and prenylation increases the molecular weight of your protein whereas proteolytic degradation decreases it. If your non-specific bands are present above your target protein you can cut them out and identify the potential PTM/s by mass spectrometry. If the non specific bands are below the target protein, then it can be proteolytic degradation. Hopefully expression of the protein at lower temperatures and purification of the protein at 4C with protease inhibitor cocktail, PMSF and benzamidine with EDTA reduces the degradation.

In addition to all of the above, it is possible that some of the additional bands are contaminating proteins. Protein purification solely by IMAC is often not sufficient to achieve a homogeneous preparation.

This may be due to the variation of conformational expression of proteins, which allows the incorporation of one or more elements of the reaction medium in 3 positions with different overall protein charges.

Looking at lines 5 and 6, I should say that you have contaminant proteins. Another step in purification process based on afinity may help.

Thanks Rosa María Martínez-Espinosa Adam B Shapiro for your answer. I have done a western blot with target protein specific antibody (see attached figD).

Saadia Karbou I have decided to do a de-glycosylation to determine this.

I agree that Fig A has a lot of higher and lower mw contaminating proteins but, judging from the description, I assume you are interested in the intense bands around your expected protein size that are very evident in 50 - 200mM elutions, FigC. While it may be possible that the non-target bands are contaminating proteins, it is unlikely that they also happen to be close to the molecular weight of your target protein. Secondly, your silver stained gel is very clean. (Fig C) No non specific bands are present besides the ones near your target protein. (50 - 200mM elutions, FigC) Also, they seem to be purified at about the same intensity as your target protein. Therefore I don't think they are contaminating proteins from the expression system. The western with the target antibody also lights up the bands suggesting that they are your target protein (fig D).

My gut feeling is that they are most likely the degradation products of your target protein. As it is a common issue with any form of heterologous protein production and purification. Hopefully the protease inhibitors and the low temperatures might reduce the amount of degradation. But the comparison of the bands with protein ladder suggests that they are of higher molecular weight than your expected protein. Nonetheless, It has been seen that some proteins can migrate a little higher/lower than the predicted molecular weight depending on their amino acid composition. But if this is not the case, your de-glycosylation assay or mass spec might shed some light.

Best

In addition to all the above, I'd like to add the possibility of incomplete cleavage of the signal sequence.

I'm guessing that you are using an alpha factor signal with Kex2 cleavage sites, and that you are secreting due to glycosylation. Depending on the protein the efficiency of the Kex2 cleavage can be sub optimal. So at least one of the bands might still have the alpha factor.

-Richard

Praveesh Valissery I did deglycosylation using NEB protocol https://www.neb.com/protocols/2016/07/29/reaction-protocols-for-protein-deglycosylation-mix-ii-p6044

The Western blot figure attached. Some band seems to disappear and the weight changes a little.