Dear Homasadat Esfahani

if you are using the SUMO tag i suppose that you perform purification after extraction from E.coli citoplasm.

if, yes In this case is not really though that the sample do not contain proteases since the E.coli citoplasm it contains proteases and if you protein contains some unfolded regions, those regions can be susceptible to protease degradation.

Do you see degradation prior IMAC (also in the loading) or only after it?

if you see degradation after IMAC i suggest to you to add some EDTA, to block at least metallo proteases and perform a second purification step asap

if you see degraration before, it means that you have degradation during the expression or lysis and you can try to reduce it by:

- change E.coli strain

- reduce expression temperature

- add protease inibitor (EDTA free during) during the cell lisis

is it yor protein contain cisteines that have to be involved in S-S bonds?

Because in this case is quite normal that you protein is not completelly folded since in the E.coli citiplasm the red-ox envitorment do not allow the formation of S-S bonds and you can try to improve the formation of it (and therefore the resistence of your protein to protease degradation) by using a different e.coli strain (eg origami) or adding an N-terminal pelB or ompA leader peptide to direct the protein expression into the citoplams

good luck

Manuele

Besides proteases:

2.1.2. Cleavage of Asp-Pro bond at 100°C—The aspartic acid-proline bond is the most susceptible bond for cleavage by heat or acidic conditions (4). The bond is cleaved by heating at high temperatures for too long. Heating at 75°C for 5 min instead of 95-100°C for 5 min has been found to avoid D-P bond cleavage, as well as completely inactivating proteases (2). However, it has to be also noted that several proteins have been found to be stable at 100°C for several hours (5).

The info was extracted from:

Article Common Artifacts and Mistakes Made in Electrophoresis

Dear Manuele Martinelli,

Thank you for your thoughtful response and for sharing your insights on protein purification,I have gained a substantial amount of knowledge from you.

I notice degradation after IMAC, but I'm concerned about whether it's advisable to use EDTA since it can chelate nickel from the resin.

and

Is it possible for nickel resin to generate strong oxidizing agents that could affect protein purification in various ways?

Thanks again for your valuable suggestions. I truly appreciate your help

Best regards,

Homa

Dear Homasadat Esfahani

of course you cannot add EDTA during the IMAC. There are some resins as the Ni-excel that are reported to be compatible with some EDTA but, my trials with those never provided good results.

You can add the EDTA just after elution to stop possible degradation process that may happen from the elution to the SDS page loading

I'm not sure if the Nickel may induce some oxidative process but in this case you can try to perform a Zn-IMAC to see if something may change but in my experience in the most of cases degradation observed in iMAC is due to the presence of proteases that during lisys and purification (expecially if this step is performed at room temperature) may induce degradation on the less strctured regions of the proteins.

best regards

Manuele

Dear Dr. Manuele Martinelli

Thank you for your valuable suggestions.

Hello Homasadat, some questions to help us troubleshoot. What is the protein of interest (POI) you are studying and what is the entire fusion (e.g., His6-SUMO-POI)? What are the sizes of your fusion protein and the ”degradation product”? Could you share the sequence of the plasmid via a SnapGene file or Word document? Does your POI oligomerize? Could you also attach an image of your polyacrylamide gel showing the column input and elite fractions? Thanks!